Journal: JBMR Plus

Article Title: Recombinant Human Bone Morphogenetic Protein 6 Delivered Within Autologous Blood Coagulum Restores Critical Size Segmental Defects of Ulna in Rabbits

doi: 10.1002/jbm4.10085

Figure Lengend Snippet: Purification and biological activity of rhBMP6. SDS‐PAGE analysis of rhBMP6: Coomassie‐stained (A) and Western blot (B). Nonreduced (37 kDa) forms are presented in lanes 1 and 6 and reduced (17 kDa) forms are in lanes 2 and 5, respectively. Molecular weight marker (ColorBurst, Sigma‐Aldrich) is indicated in lanes 3 and 4, respectively. (C) Specific rhBMP6 activity in C2C12‐BRE‐Luc reporter gene assay: comparison between two different clinical batches. The activity was tested in a range of concentrations as in x axis and expressed in relative light units (RLU) as in y axis. Data represent mean ± SEM of 3 independent measurements.

Article Snippet: The liquid portion (serum) was removed and the homogeneous, cohesive, injectable and malleable ABGS gel was ready for use. rhBMP6 characterization and release studies For rhBMP6 identification and characterization, lyophilized protein samples were subjected to SDS‐PAGE electrophoresis, transferred to the nitrocellulose membrane, and analyzed by Western blot using the rhBMP6‐specific monoclonal antibody (available in the rhBMP6 DuoSet ELISA kit, R&D Systems, DY507).

Techniques: Purification, Activity Assay, SDS Page, Staining, Western Blot, Molecular Weight, Marker, Reporter Gene Assay, Comparison

Journal: JBMR Plus

Article Title: Recombinant Human Bone Morphogenetic Protein 6 Delivered Within Autologous Blood Coagulum Restores Critical Size Segmental Defects of Ulna in Rabbits

doi: 10.1002/jbm4.10085

Figure Lengend Snippet: Characterization of coagulation and binding, release, and stability of rhBMP6 in autologous bone graft substitute (ABGS). (A) Cumulative frequency of coagulation formation over time. Raw data (n/N) and percentages are shown. Bracketed values are exact 95% confidence intervals (left‐sided 97.5% at 60 minutes). (B) Release profile of rhBMP6 from ABGS using two different concentrations of rhBMP6 over a 14‐day period, as determined by ELISA. Data represent mean ± SEM of 3 independent measurements. (C) The stability of rhBMP6 during preparation of the ABGS implant was maintained over 60 minutes (lane 4) and 90 minutes (lane 5) with a loss of around 5% during preparation (lane 3). Arrow indicates ∼35 kDa band, which corresponds to the mature rhBMP6 under nonreduced condition. (D) Semiquantitative analysis of 99mTc‐labeled rhBMP6 binding to blood components in a dot blot assay. Mean + SEM (n = 3) are shown.

Article Snippet: The liquid portion (serum) was removed and the homogeneous, cohesive, injectable and malleable ABGS gel was ready for use. rhBMP6 characterization and release studies For rhBMP6 identification and characterization, lyophilized protein samples were subjected to SDS‐PAGE electrophoresis, transferred to the nitrocellulose membrane, and analyzed by Western blot using the rhBMP6‐specific monoclonal antibody (available in the rhBMP6 DuoSet ELISA kit, R&D Systems, DY507).

Techniques: Coagulation, Binding Assay, Enzyme-linked Immunosorbent Assay, Labeling, Dot Blot

Journal: JBMR Plus

Article Title: Recombinant Human Bone Morphogenetic Protein 6 Delivered Within Autologous Blood Coagulum Restores Critical Size Segmental Defects of Ulna in Rabbits

doi: 10.1002/jbm4.10085

Figure Lengend Snippet: Evaluation of ABGS in rat subcutaneous implants harvested at days 1, 3, 7, and 35. (A) Photomicrographs of histology of ABC alone at days 1, 3, and 7. Size marker: left column 500 μm (magnification 4×); middle column 200 μm (magnification 10×); right column upper (top) image 20 μm (magnification 60×) and middle and lower (bottom) image 50 μm (magnification (40×). (B) Photomicrographs of histology of ABGS (25 μg rhBMP6 per implant) at days 1, 3, 7, and 35. Size marker: left column 500 μm (magnification 4×); middle column upper (top) and lower (bottom) image 200 μm (magnification 10×), middle images 500 μm and 200 μm (magnification 4× and 10×, respectively); right column upper (top) image 20 μm (magnification 60×) and middle and lower (bottom) images 50 μm (magnification 40×). Asterisks denote clearly demarcated zone composed of osteoprogenitor cells stained positive for alkaline phosphatase on day 1; with condensations of extracellular matrix (black arrows) and formation of early chondrocytes within the osteoprogenitor zone (black arrowheads). On day 3, cells from outside the ABC slowly penetrated and hypertrophic chondrocytes in endochondral bone formation appeared on day 7 (yellow arrowheads). On day 35, dense trabecular bone (yellow arrows) with a broad cortexlike structure from outside demonstrated a solid persistent bone ossicle (green arrowhead). (C) The neutrophil infiltration on histological sections. The implants were examined on day 3 after implantation of 20 μg rhBMP2/300 mg bovine Achilles tendon, 20 μg rhBMP7/300 mg bovine bone collagen carrier, or 20 μg rhBMP6/300 μL ABC, respectively. Mean ± SEM (n = 10), *p < 0.01 versus rhBMP6, **p < 0.05 versus rhBMP6. (D) Myeloperoxidase (MPO) activity. Mean ± SEM (n = 8), *p < 0.01 versus rhBMP6, **p < 0.05 versus rhBMP6.

Article Snippet: The liquid portion (serum) was removed and the homogeneous, cohesive, injectable and malleable ABGS gel was ready for use. rhBMP6 characterization and release studies For rhBMP6 identification and characterization, lyophilized protein samples were subjected to SDS‐PAGE electrophoresis, transferred to the nitrocellulose membrane, and analyzed by Western blot using the rhBMP6‐specific monoclonal antibody (available in the rhBMP6 DuoSet ELISA kit, R&D Systems, DY507).

Techniques: Marker, Staining, Activity Assay

Journal: JBMR Plus

Article Title: Recombinant Human Bone Morphogenetic Protein 6 Delivered Within Autologous Blood Coagulum Restores Critical Size Segmental Defects of Ulna in Rabbits

doi: 10.1002/jbm4.10085

Figure Lengend Snippet: Rat subcutaneous implants. (A) Rat subcutaneous implants in vivo. The white arrow indicates the implant; white circle shows lack of fibrotic tissue accumulation. (B) Bone volume (BV) calculated after micro‐CT scan in rat subcutaneous implants at day 35 with various rhBMP6 doses and accompanying 3D models of the newly formed bone. The rhBMP6 dose used is represented as μg/implant. Mean ± SEM (n = 4 per dose), *p < 0.01 versus 2.5 μg, 5 μg, and 10 μg; **p < 0.05 versus 2.5 μg.

Article Snippet: The liquid portion (serum) was removed and the homogeneous, cohesive, injectable and malleable ABGS gel was ready for use. rhBMP6 characterization and release studies For rhBMP6 identification and characterization, lyophilized protein samples were subjected to SDS‐PAGE electrophoresis, transferred to the nitrocellulose membrane, and analyzed by Western blot using the rhBMP6‐specific monoclonal antibody (available in the rhBMP6 DuoSet ELISA kit, R&D Systems, DY507).

Techniques: In Vivo, Micro-CT

Journal: JBMR Plus

Article Title: Recombinant Human Bone Morphogenetic Protein 6 Delivered Within Autologous Blood Coagulum Restores Critical Size Segmental Defects of Ulna in Rabbits

doi: 10.1002/jbm4.10085

Figure Lengend Snippet: Radiographs of bone healing through the course of 0 to 23 weeks for different doses of rhBMP6 (0, 25, 50 and 100 µg). Representative rabbit from each treatment group is presented.

Article Snippet: The liquid portion (serum) was removed and the homogeneous, cohesive, injectable and malleable ABGS gel was ready for use. rhBMP6 characterization and release studies For rhBMP6 identification and characterization, lyophilized protein samples were subjected to SDS‐PAGE electrophoresis, transferred to the nitrocellulose membrane, and analyzed by Western blot using the rhBMP6‐specific monoclonal antibody (available in the rhBMP6 DuoSet ELISA kit, R&D Systems, DY507).

Techniques:

Journal: JBMR Plus

Article Title: Recombinant Human Bone Morphogenetic Protein 6 Delivered Within Autologous Blood Coagulum Restores Critical Size Segmental Defects of Ulna in Rabbits

doi: 10.1002/jbm4.10085

Figure Lengend Snippet: The effect of rhBMP6 dose on bone healing in rabbit ulna segmental defect. (A) Radiographs of all bone samples (n = 5 per group) at week 23. (B) Radiographic grading scores (0–6) of all bone samples using an established scoring system.38, 39 (C) 3D models (longitudinal and cross‐sectional) of bone healing in rabbits after ex vivo micro‐CT scan at week 23 after study termination. Three bone samples per dose group are shown, with corresponding X‐ray image on the left. Yellow arrows indicate initial defect size and introduced bone osteotomy sites. White asterisk indicates a partial synostosis between ulna and radius. (D) Quantitative analysis of newly formed bone revealed a dose‐dependent manner of rhBMP6 stimulation of bone defect healing. For quantification, the defect site was approximated and delineated manually after which parameters for bone volume (BV) and newly formed endosteal/medullary volume (MV) were calculated. 3D models of the scanned bones were obtained using CTVox (Bruker) software. Quantitative micro‐CT parameters were analyzed by one‐way ANOVA with post hoc test for linear trend. Each bar represents mean ± SD.

Article Snippet: The liquid portion (serum) was removed and the homogeneous, cohesive, injectable and malleable ABGS gel was ready for use. rhBMP6 characterization and release studies For rhBMP6 identification and characterization, lyophilized protein samples were subjected to SDS‐PAGE electrophoresis, transferred to the nitrocellulose membrane, and analyzed by Western blot using the rhBMP6‐specific monoclonal antibody (available in the rhBMP6 DuoSet ELISA kit, R&D Systems, DY507).

Techniques: Ex Vivo, Micro-CT, Software

Journal: JBMR Plus

Article Title: Recombinant Human Bone Morphogenetic Protein 6 Delivered Within Autologous Blood Coagulum Restores Critical Size Segmental Defects of Ulna in Rabbits

doi: 10.1002/jbm4.10085

Figure Lengend Snippet: Evaluation ABGS in ulna critical size defect in rabbits. (A) Photomicrographs of histology of representative slides from animals treated with rhBMP6. Rabbits treated with 100 μg rhBMP6 showed a complete restoration with cortical and trabecular bone in the diaphysis and rabbits treated with 50 μg rhBMP6 showed a complete healing with delayed remodeling, while rabbits treated with 25 μg rhBMP6 showed an incomplete rebridgement of bone defect showing a dose‐dependent effect at 23 weeks after surgery. Yellow arrowheads denote the size of surgical defects. (B) Magnified section of newly formed bone from yellow rectangles in A. Black arrowheads indicate blood vessels, and arrows indicate enlarged osteocyte lacunae in the new woven bone under remodeling. Size marker indicates 200 μm.

Article Snippet: The liquid portion (serum) was removed and the homogeneous, cohesive, injectable and malleable ABGS gel was ready for use. rhBMP6 characterization and release studies For rhBMP6 identification and characterization, lyophilized protein samples were subjected to SDS‐PAGE electrophoresis, transferred to the nitrocellulose membrane, and analyzed by Western blot using the rhBMP6‐specific monoclonal antibody (available in the rhBMP6 DuoSet ELISA kit, R&D Systems, DY507).

Techniques: Marker

Journal: JBMR Plus

Article Title: Recombinant Human Bone Morphogenetic Protein 6 Delivered Within Autologous Blood Coagulum Restores Critical Size Segmental Defects of Ulna in Rabbits

doi: 10.1002/jbm4.10085

Figure Lengend Snippet: (A) Comparison of rhBMP7/bovine bone collagen with ABGS implant (rhBMP6/ABC) (right) in rabbit ulna segmental defect models at 2 and 6 weeks after administration. (B) In vivo X‐ray analysis of critical size defects of rabbit ulna 2 and 6 weeks after surgery, treated with 100 μg BMP7 + bovine bone collagen, 100 μg of BMP6 + ABC, and a bovine collagen alone (control) (n = 8 rabbits per group). *p < 0.05 versus BMP7; **p < 0.01 versus control (ANOVA, Dunnett test). (C) Ex vivo micro‐CT analysis of critical size defects of rabbit ulnas 8 weeks after treatment (the same as in B, n = 8 per group). *p < 0.05 versus BMP7; **p < 0.01 versus control (ANOVA, Dunnett test). (D) Effects of the inhibitory action of Noggin as examined for BRE‐driven luciferase activity in C2C12 cells for BMP6 and BMP7. Each data point is mean ± SD of 3 measured values. *p < 0.01 versus BMP6.

Article Snippet: The liquid portion (serum) was removed and the homogeneous, cohesive, injectable and malleable ABGS gel was ready for use. rhBMP6 characterization and release studies For rhBMP6 identification and characterization, lyophilized protein samples were subjected to SDS‐PAGE electrophoresis, transferred to the nitrocellulose membrane, and analyzed by Western blot using the rhBMP6‐specific monoclonal antibody (available in the rhBMP6 DuoSet ELISA kit, R&D Systems, DY507).

Techniques: Comparison, In Vivo, Control, Ex Vivo, Micro-CT, Luciferase, Activity Assay

Journal: PLoS ONE

Article Title: The BMP Pathway Participates in Human Naive CD4 + T Cell Activation and Homeostasis

doi: 10.1371/journal.pone.0131453

Figure Lengend Snippet: Freshly isolated human peripheral blood naive CD4 + T cells were stimulated with anti-CD3/CD28 mAb. (A) Transcripts for several components of the canonical BMP signaling pathway were determined by real-time PCR ex vivo (0h) or after 6 days of stimulation (6d). GNB2L1 was used as endogenous control. Means ± SD of at least three independent experiments run in duplicates are shown. Note the logarithmic scale on y-axis. (B) Percentage of BMPRIA + cells detected by flow cytometry throughout the culture. Bars represent the mean ± SD of two to five independent experiments. (C) Expression of BMPRIA and CD25 in T cells (upper dot plots) and differential expression of BMPRIA in the CD25 - and CD25 + cell populations (lower histograms) during activation. A representative experiment out of four is shown. (D) Expression of BMPRIA in T cells cultured with different stimuli. Grey histograms represent isotype controls. Similar stainings were obtained in two to three independent experiments. mDCs: mature dendritic cells; PHA: Phytohaemagglutinin; Ck: cytokine cocktail (rhIL-2, rhIL-6, rhTNF-α) (E) Determination of BMP2/4 and BMP6 production by flow cytometry in T cells cultured in media alone (grey histograms) or in the presence of anti-CD3/CD28 mAb (black histograms). Grey filled histograms represent isotype control stainings. A representative experiment out of three is shown. (F) Expression of CD25 and phosphorylated BR-Smad (pBR-Smads) during activation. For comparison, T cells were kept in culture media alone. Results are representative of three independent experiments.

Article Snippet: For the intracellular stainings, and according to the manufacturer’s instructions, cells were treated with Cytofix/Cytoperm solution (BD Biosciences) for 20 min at 4°C, washed with Perm/Wash buffer (BD Biosciences), and stained with purified anti-human BMP2/4 mAb (100230), biotin-conjugated anti-human BMP6, purified anti-human BMPRIA (all from R&D Systems) or PE-conjugated anti-human Bcl-2 mAb (6C8) (BD Biosciences), followed when required by fluorochrome-conjugated, multiadsorbed F(ab’)2 fragments of donkey anti-goat, anti-rabbit or anti-mouse IgG or streptavidin (Jackson ImmunoResearch Laboratories), all diluted in Perm/Wash buffer.

Techniques: Isolation, Real-time Polymerase Chain Reaction, Ex Vivo, Flow Cytometry, Expressing, Activation Assay, Cell Culture, Comparison

Journal: PLoS ONE

Article Title: The BMP Pathway Participates in Human Naive CD4 + T Cell Activation and Homeostasis

doi: 10.1371/journal.pone.0131453

Figure Lengend Snippet: (A) Expression of BMP2/4 and BMP6 was determined by flow cytometry at the indicated time points in T cells cultured in media alone (grey histograms) or in the presence of IL-7 (5 ng/ml) (black histograms). Grey filled histograms represent isotype control stainings. A representative experiment out of four is shown. (B) Differential expression of phosphorylated Smad-1/5/8 (pBR-Smad) analyzed by flow cytometry in naive CD4 + T cells after 11 days of culture in media alone or supplemented with IL-7 and DMSO or IL-7 and DMH1 (40 μM). Percentages represent the increment relative to cultures in media alone. One representative of three independent experiments is shown.

Article Snippet: For the intracellular stainings, and according to the manufacturer’s instructions, cells were treated with Cytofix/Cytoperm solution (BD Biosciences) for 20 min at 4°C, washed with Perm/Wash buffer (BD Biosciences), and stained with purified anti-human BMP2/4 mAb (100230), biotin-conjugated anti-human BMP6, purified anti-human BMPRIA (all from R&D Systems) or PE-conjugated anti-human Bcl-2 mAb (6C8) (BD Biosciences), followed when required by fluorochrome-conjugated, multiadsorbed F(ab’)2 fragments of donkey anti-goat, anti-rabbit or anti-mouse IgG or streptavidin (Jackson ImmunoResearch Laboratories), all diluted in Perm/Wash buffer.

Techniques: Expressing, Flow Cytometry, Cell Culture