Journal: bioRxiv

Article Title: Bioactive coatings on 3D printed scaffolds for bone regeneration: Translation from in vitro to in vivo models and the impact of material properties and growth factor concentration

doi: 10.1101/2023.10.22.560309

Figure Lengend Snippet: HBMSC viability and live (green)/dead (red) cell labelling on uncoated, ELP, PEA/FN/BMP-2 and ELP/PEA/FN/BMP-2 coated PCL-TMA scaffolds. (A) alamarBlue™ HS fluorescence results of ELP, PEA/FN/BMP-2 and ELP/PEA/FN/BMP-2 coated PCL-TMA scaffolds show no significant difference between uncoated PCL-TMA and coated scaffolds, however all coating variations showed a significantly increased fluorescence result at day 14 compared to uncoated PCL-TMA. 2-way ANOVA with Dunnett’s multiple comparisons test, n=3, mean and S.D. shown, ns; non-significant, ****p<0.001. (B) The cell number and location of cells adhered to the scaffold initially at day 1 and subsequent increase in cell coverage at day 14, as seen by fluorescent labelling of cells. Scale bar 1 mm.

Article Snippet: Reagents were purchased as follows: Ethyl acrylate (Sigma, UK), ELP with statherin sequence (SN A 15) (Technical Proteins Nanobiotechnology, Valladolid, Spain); collagenase (Gibco, UK); human fibronectin and human recombinant BMP-2 (R&D systems, Biotechne, UK); recombinant human BMP-2 (Infuse/InductOS® Bone graft kit, Medtronic, USA); alcian blue 8X, light green SF, orange G 85% pure, Paraformaldehyde 96% extra pure, phosphomolybdic acid hydrate 80% (Acros Organics); Picrosirius Red, Van Gieson’s stain, Weigert’s Haematoxylin Parts 1 and 2 (Clintech Ltd, UK); Benzoyl peroxide, GMA solution B, JB4 solution A (Polysciences); GoTaq qPCR master mix, Herring sperm DNA, RNeasy mini prep RNA extraction kit (Promega); phosphate buffered saline (PBS), trypsin/ ethylenediaminetetraacetic acid (EDTA), Dulbecco’s Modified Eagle Medium (DMEM), Alpha Minimum Essential Medium (αMEM), penicillin-streptomycin (Scientific Laboratory Supplies, SLS); 4-Nitrophenol solution 10 nM, acetic acid, acetone, acid fushsin, alizarin red S, alkaline buffer solution, ascorbic acid-2-phosphate, beta-glycerophosphate disodium hydrate salt (βGP), cell lytic M, dexamethasone, fast violet B salts, glycine, histowax, hydrochloric acid, iodoacetamide, ipegal, L-glutamic acid, Naphthol AS-MX phosphate 0.25%, parafilm, PBS (with CaCl 2 /MgCl 2 ), phenyl methyl sulphonyl fluoride, phosphatase substrate, polysorbate 80, ponceau xylidine, silver nitrate, sodium chloride, sodium hydroxide pellets, sucrose, TRIS-EDTA (TE) buffer solution (Merck, UK); Embedding capsule (TAAB Laboratories equipment); alamarBlue™ HS Cell Viability Reagent, 70 µM cell strainer, dibutyl phthalate xylene (DPX), ethidium homodimer-1, fetal calf serum (FCS), fisherbrand grade 01 cellulose general purpose filter paper, Histoclear, isopropanol, methyl benzoate, Quanti-IT™ Picogreen™ ds DNA reagent, Taqman® Reverse Transcription Kit, Vybrant™ CFDA SE Cell Tracer Kit (Thermofisher Scientific, UK); Fast green and sodium thiosulphate (VWR); Lubrithal (Dechra, UK), Isoflurane (Dechra, UK), Buprenorphine (Buprecare® multidose, Animalcare, UK) and Vetasept® sourced from MWI animal health, UK.

Techniques: Fluorescence

Journal: bioRxiv

Article Title: Bioactive coatings on 3D printed scaffolds for bone regeneration: Translation from in vitro to in vivo models and the impact of material properties and growth factor concentration

doi: 10.1101/2023.10.22.560309

Figure Lengend Snippet: Assessment of HBMSC differentiation on coated 3D scaffold materials. (A) ALP specific activity of HBMSCs on ELP, PEA/FN/BMP-2 and ELP/PEA/FN/BMP-2 coated PCL-TMA scaffolds at day 7. There was no significant difference between the uncoated PCL-TMA and the coatings of interest in basal or osteogenic conditions. (B) ALP specific activity results comparing 100 ng/mL and 5 µg/mL BMP-2 coating solution concentration at day 7. There was a significant increase in ALP production by HBMSCs in response to the 5 μg/mL BMP-2 coating solution compared to the 100 ng/mL concentration BMP-2 solution in osteogenic culture conditions only. (C) ALP gene expression at day 7 was significantly enhanced for ELP coating than uncoated nylon in basal culture conditions, with the PEA/FN/BMP-2, ELP/PEA/FN/BMP-2 coatings displaying significantly reduced ALP gene expression in osteogenic conditions. (D) Collagen1A1 gene expression at day 7 was not significantly greater than uncoated nylon for any of the coatings in basal or osteogenic media conditions. 2-way ANOVA with Dunnett’s multiple comparisons test, n=3, mean and S.D. shown, ns; non-significant, *p<0.05, **p<0.01, ****p=<0.0001. (E) Alizarin red staining at day 27 indicated red stain uptake in the ELP coated and mineralised scaffolds when no cells were seeded, due to the constituents of the coatings themselves. ELP coating alone and with subsequent mineralisation, lead to enhanced staining due to mineral deposition on the surface of the scaffold in osteogenic culture conditions. Representative images shown, n=3, scale bar 1 mm.

Article Snippet: Reagents were purchased as follows: Ethyl acrylate (Sigma, UK), ELP with statherin sequence (SN A 15) (Technical Proteins Nanobiotechnology, Valladolid, Spain); collagenase (Gibco, UK); human fibronectin and human recombinant BMP-2 (R&D systems, Biotechne, UK); recombinant human BMP-2 (Infuse/InductOS® Bone graft kit, Medtronic, USA); alcian blue 8X, light green SF, orange G 85% pure, Paraformaldehyde 96% extra pure, phosphomolybdic acid hydrate 80% (Acros Organics); Picrosirius Red, Van Gieson’s stain, Weigert’s Haematoxylin Parts 1 and 2 (Clintech Ltd, UK); Benzoyl peroxide, GMA solution B, JB4 solution A (Polysciences); GoTaq qPCR master mix, Herring sperm DNA, RNeasy mini prep RNA extraction kit (Promega); phosphate buffered saline (PBS), trypsin/ ethylenediaminetetraacetic acid (EDTA), Dulbecco’s Modified Eagle Medium (DMEM), Alpha Minimum Essential Medium (αMEM), penicillin-streptomycin (Scientific Laboratory Supplies, SLS); 4-Nitrophenol solution 10 nM, acetic acid, acetone, acid fushsin, alizarin red S, alkaline buffer solution, ascorbic acid-2-phosphate, beta-glycerophosphate disodium hydrate salt (βGP), cell lytic M, dexamethasone, fast violet B salts, glycine, histowax, hydrochloric acid, iodoacetamide, ipegal, L-glutamic acid, Naphthol AS-MX phosphate 0.25%, parafilm, PBS (with CaCl 2 /MgCl 2 ), phenyl methyl sulphonyl fluoride, phosphatase substrate, polysorbate 80, ponceau xylidine, silver nitrate, sodium chloride, sodium hydroxide pellets, sucrose, TRIS-EDTA (TE) buffer solution (Merck, UK); Embedding capsule (TAAB Laboratories equipment); alamarBlue™ HS Cell Viability Reagent, 70 µM cell strainer, dibutyl phthalate xylene (DPX), ethidium homodimer-1, fetal calf serum (FCS), fisherbrand grade 01 cellulose general purpose filter paper, Histoclear, isopropanol, methyl benzoate, Quanti-IT™ Picogreen™ ds DNA reagent, Taqman® Reverse Transcription Kit, Vybrant™ CFDA SE Cell Tracer Kit (Thermofisher Scientific, UK); Fast green and sodium thiosulphate (VWR); Lubrithal (Dechra, UK), Isoflurane (Dechra, UK), Buprenorphine (Buprecare® multidose, Animalcare, UK) and Vetasept® sourced from MWI animal health, UK.

Techniques: Activity Assay, Concentration Assay, Expressing, Staining

Journal: bioRxiv

Article Title: Bioactive coatings on 3D printed scaffolds for bone regeneration: Translation from in vitro to in vivo models and the impact of material properties and growth factor concentration

doi: 10.1101/2023.10.22.560309

Figure Lengend Snippet: CAM assay viability and Chalkley score results for PCL-TMA scaffolds. (A) Chick viability was suboptimal due to poor chick development, n=6. (B) There was no significant difference in Chalkley score between uncoated PCL-TMA and the coated scaffolds, (uncoated n=4, ELP n=4, PEA/FN/BMP-2 n=4, ELP/PEA/FN/BMP-2 n=3), ns=non-significant. One-way ANOVA with Dunnett’s multiple comparisons test was used for statistical analysis, mean and S.D. shown. (C) Photographs of representative uncoated PCL-TMA and ELP coating, PEA/FN/BMP-2 and ELP/PEA/FN/BMP-2 coated PCL-TMA scaffolds on the CAM. The PCL-TMA material, the ELP and PEA/FN/BMP-2 coatings were biocompatible and supported angiogenesis. Scale bar 5 mm. (D) Histological staining (Alcian blue and Sirius red or Goldner’s trichrome) of PCL-TMA scaffolds surrounded by CAM tissue. The PCL-TMA scaffold material did not support sectioning, with fragments remaining (arrow), but tissue around the prior scaffold (*) could be determined. Scale bar 100 μm.

Article Snippet: Reagents were purchased as follows: Ethyl acrylate (Sigma, UK), ELP with statherin sequence (SN A 15) (Technical Proteins Nanobiotechnology, Valladolid, Spain); collagenase (Gibco, UK); human fibronectin and human recombinant BMP-2 (R&D systems, Biotechne, UK); recombinant human BMP-2 (Infuse/InductOS® Bone graft kit, Medtronic, USA); alcian blue 8X, light green SF, orange G 85% pure, Paraformaldehyde 96% extra pure, phosphomolybdic acid hydrate 80% (Acros Organics); Picrosirius Red, Van Gieson’s stain, Weigert’s Haematoxylin Parts 1 and 2 (Clintech Ltd, UK); Benzoyl peroxide, GMA solution B, JB4 solution A (Polysciences); GoTaq qPCR master mix, Herring sperm DNA, RNeasy mini prep RNA extraction kit (Promega); phosphate buffered saline (PBS), trypsin/ ethylenediaminetetraacetic acid (EDTA), Dulbecco’s Modified Eagle Medium (DMEM), Alpha Minimum Essential Medium (αMEM), penicillin-streptomycin (Scientific Laboratory Supplies, SLS); 4-Nitrophenol solution 10 nM, acetic acid, acetone, acid fushsin, alizarin red S, alkaline buffer solution, ascorbic acid-2-phosphate, beta-glycerophosphate disodium hydrate salt (βGP), cell lytic M, dexamethasone, fast violet B salts, glycine, histowax, hydrochloric acid, iodoacetamide, ipegal, L-glutamic acid, Naphthol AS-MX phosphate 0.25%, parafilm, PBS (with CaCl 2 /MgCl 2 ), phenyl methyl sulphonyl fluoride, phosphatase substrate, polysorbate 80, ponceau xylidine, silver nitrate, sodium chloride, sodium hydroxide pellets, sucrose, TRIS-EDTA (TE) buffer solution (Merck, UK); Embedding capsule (TAAB Laboratories equipment); alamarBlue™ HS Cell Viability Reagent, 70 µM cell strainer, dibutyl phthalate xylene (DPX), ethidium homodimer-1, fetal calf serum (FCS), fisherbrand grade 01 cellulose general purpose filter paper, Histoclear, isopropanol, methyl benzoate, Quanti-IT™ Picogreen™ ds DNA reagent, Taqman® Reverse Transcription Kit, Vybrant™ CFDA SE Cell Tracer Kit (Thermofisher Scientific, UK); Fast green and sodium thiosulphate (VWR); Lubrithal (Dechra, UK), Isoflurane (Dechra, UK), Buprenorphine (Buprecare® multidose, Animalcare, UK) and Vetasept® sourced from MWI animal health, UK.

Techniques: Chick Chorioallantoic Membrane Assay, Staining

Journal: bioRxiv

Article Title: Bioactive coatings on 3D printed scaffolds for bone regeneration: Translation from in vitro to in vivo models and the impact of material properties and growth factor concentration

doi: 10.1101/2023.10.22.560309

Figure Lengend Snippet: µCT results of the murine subcutaneous implantation study. (A) Representative µCT images with no bone formation observed in uncoated, PEA/FN/BMP-2, ELP, or ELP/PEA/FN/BMP-2 coated PCL-TMA scaffolds, however the collagen sponge with 5 µg of BMP-2 showed mineralisation in all mice (within red circles) at week 6. (B) Quantification of bone volume formed in the mouse subcutaneous implant model using PCL-TMA scaffolds and collagen sponge/BMP-2. (i) The collagen sponge displayed significant bone formation at 8 weeks compared to the uncoated PCL-TMA scaffold. (ii) There was no significant difference between the negligible bone formation on the coated scaffolds compared to the uncoated PCL-TMA scaffolds. One-way ANOVA with Dunnett’s multiple comparisons test, ns= not significant, ****p<0.0001. N=9 uncoated scaffolds, n=9 collagen sponge, n=6 PEA/FN/BMP-2 and n=6 ELP/PEA/FN/BMP-2 coated scaffolds, mean and S.D. shown.

Article Snippet: Reagents were purchased as follows: Ethyl acrylate (Sigma, UK), ELP with statherin sequence (SN A 15) (Technical Proteins Nanobiotechnology, Valladolid, Spain); collagenase (Gibco, UK); human fibronectin and human recombinant BMP-2 (R&D systems, Biotechne, UK); recombinant human BMP-2 (Infuse/InductOS® Bone graft kit, Medtronic, USA); alcian blue 8X, light green SF, orange G 85% pure, Paraformaldehyde 96% extra pure, phosphomolybdic acid hydrate 80% (Acros Organics); Picrosirius Red, Van Gieson’s stain, Weigert’s Haematoxylin Parts 1 and 2 (Clintech Ltd, UK); Benzoyl peroxide, GMA solution B, JB4 solution A (Polysciences); GoTaq qPCR master mix, Herring sperm DNA, RNeasy mini prep RNA extraction kit (Promega); phosphate buffered saline (PBS), trypsin/ ethylenediaminetetraacetic acid (EDTA), Dulbecco’s Modified Eagle Medium (DMEM), Alpha Minimum Essential Medium (αMEM), penicillin-streptomycin (Scientific Laboratory Supplies, SLS); 4-Nitrophenol solution 10 nM, acetic acid, acetone, acid fushsin, alizarin red S, alkaline buffer solution, ascorbic acid-2-phosphate, beta-glycerophosphate disodium hydrate salt (βGP), cell lytic M, dexamethasone, fast violet B salts, glycine, histowax, hydrochloric acid, iodoacetamide, ipegal, L-glutamic acid, Naphthol AS-MX phosphate 0.25%, parafilm, PBS (with CaCl 2 /MgCl 2 ), phenyl methyl sulphonyl fluoride, phosphatase substrate, polysorbate 80, ponceau xylidine, silver nitrate, sodium chloride, sodium hydroxide pellets, sucrose, TRIS-EDTA (TE) buffer solution (Merck, UK); Embedding capsule (TAAB Laboratories equipment); alamarBlue™ HS Cell Viability Reagent, 70 µM cell strainer, dibutyl phthalate xylene (DPX), ethidium homodimer-1, fetal calf serum (FCS), fisherbrand grade 01 cellulose general purpose filter paper, Histoclear, isopropanol, methyl benzoate, Quanti-IT™ Picogreen™ ds DNA reagent, Taqman® Reverse Transcription Kit, Vybrant™ CFDA SE Cell Tracer Kit (Thermofisher Scientific, UK); Fast green and sodium thiosulphate (VWR); Lubrithal (Dechra, UK), Isoflurane (Dechra, UK), Buprenorphine (Buprecare® multidose, Animalcare, UK) and Vetasept® sourced from MWI animal health, UK.

Techniques:

Journal: bioRxiv

Article Title: Bioactive coatings on 3D printed scaffolds for bone regeneration: Translation from in vitro to in vivo models and the impact of material properties and growth factor concentration

doi: 10.1101/2023.10.22.560309

Figure Lengend Snippet: Alcian blue and Sirius red, Goldner’s trichrome, Alizarin red and Von Kossa staining of PCL-TMA scaffolds and collagen sponge/BMP-2. The scaffolds were not amenable to sectioning; however, the surrounding tissue remained. (*) PCL-TMA scaffold area or collagen sponge/BMP-2. (A) Shards of PCL-TMA material (black arrow) remain in the section. (B) Vivid red staining muscle was seen but no bone formation. (C and D) No bone formation was found on the uncoated scaffold. (E - H) Only the collagen sponge displayed marked mineralisation and bone formation around the periphery (arrows). (I - T) No bone formation was seen on the ELP, PEA/FN/BMP-2 or ELP/PEA/FN/BMP-2 coated scaffolds, with the ridges of the scaffold material seen surrounded by tissue (J arrow). Scale bar 100 μm.

Article Snippet: Reagents were purchased as follows: Ethyl acrylate (Sigma, UK), ELP with statherin sequence (SN A 15) (Technical Proteins Nanobiotechnology, Valladolid, Spain); collagenase (Gibco, UK); human fibronectin and human recombinant BMP-2 (R&D systems, Biotechne, UK); recombinant human BMP-2 (Infuse/InductOS® Bone graft kit, Medtronic, USA); alcian blue 8X, light green SF, orange G 85% pure, Paraformaldehyde 96% extra pure, phosphomolybdic acid hydrate 80% (Acros Organics); Picrosirius Red, Van Gieson’s stain, Weigert’s Haematoxylin Parts 1 and 2 (Clintech Ltd, UK); Benzoyl peroxide, GMA solution B, JB4 solution A (Polysciences); GoTaq qPCR master mix, Herring sperm DNA, RNeasy mini prep RNA extraction kit (Promega); phosphate buffered saline (PBS), trypsin/ ethylenediaminetetraacetic acid (EDTA), Dulbecco’s Modified Eagle Medium (DMEM), Alpha Minimum Essential Medium (αMEM), penicillin-streptomycin (Scientific Laboratory Supplies, SLS); 4-Nitrophenol solution 10 nM, acetic acid, acetone, acid fushsin, alizarin red S, alkaline buffer solution, ascorbic acid-2-phosphate, beta-glycerophosphate disodium hydrate salt (βGP), cell lytic M, dexamethasone, fast violet B salts, glycine, histowax, hydrochloric acid, iodoacetamide, ipegal, L-glutamic acid, Naphthol AS-MX phosphate 0.25%, parafilm, PBS (with CaCl 2 /MgCl 2 ), phenyl methyl sulphonyl fluoride, phosphatase substrate, polysorbate 80, ponceau xylidine, silver nitrate, sodium chloride, sodium hydroxide pellets, sucrose, TRIS-EDTA (TE) buffer solution (Merck, UK); Embedding capsule (TAAB Laboratories equipment); alamarBlue™ HS Cell Viability Reagent, 70 µM cell strainer, dibutyl phthalate xylene (DPX), ethidium homodimer-1, fetal calf serum (FCS), fisherbrand grade 01 cellulose general purpose filter paper, Histoclear, isopropanol, methyl benzoate, Quanti-IT™ Picogreen™ ds DNA reagent, Taqman® Reverse Transcription Kit, Vybrant™ CFDA SE Cell Tracer Kit (Thermofisher Scientific, UK); Fast green and sodium thiosulphate (VWR); Lubrithal (Dechra, UK), Isoflurane (Dechra, UK), Buprenorphine (Buprecare® multidose, Animalcare, UK) and Vetasept® sourced from MWI animal health, UK.

Techniques: Staining

Journal: Experimental and Therapeutic Medicine

Article Title: Triptolide inhibits the function of TNF-α in osteoblast differentiation by inhibiting the NF-κB signaling pathway

doi: 10.3892/etm.2017.4749

Figure Lengend Snippet: TNF-α significantly inhibited the BMP-2-induced osteoblast differentiation. (A) Effect of BMP-2 on osteogenic activity. C2C12 cells were treated with 200 ng/ml BMP-2 for 3 days and the ALP activity was assessed. **P<0.01 vs. Ctrl group. (B) Effect of TNF-α on osteoblast activity. C2C12 cells were treated with 5 ng/ml TNF-α for 3 days and the ALP activity was determined. *P<0.05 vs. Ctrl group. (C) TNF-α inhibited the effect of BMP-2 on osteogenic activity. C2C12 cells were treated with 200 ng/ml BMP-2 or 200 ng/ml BMP + 5 ng/ml TNF-α for 3 days and the ALP activity was determined. ##P<0.01 vs. Ctrl group, **P<0.01 vs. BMP-2 group. TNF, tumor necrosis factor; BMP, bone morphogenetic protein; ALP, alkaline phosphatase; Ctrl, control.

Article Snippet: In the present study, C2C12 cells were treated with BMP-2 (10426-H01H-10; Sinobiological, Inc., Beijing, China) in order to produce osteoblast cells.

Techniques: Activity Assay

Journal: Experimental and Therapeutic Medicine

Article Title: Triptolide inhibits the function of TNF-α in osteoblast differentiation by inhibiting the NF-κB signaling pathway

doi: 10.3892/etm.2017.4749

Figure Lengend Snippet: TNF-α inhibited BMP-2-induced osteoblast differentiation in a dose-dependent manner. (A) C2C12 cells were treated with 100, 200 or 300 ng/ml BMP-2 for 3 days and ALP activity was evaluated. **P<0.01 vs. untreated cells. (B) C2C12 cells were treated with 100 ng/ml BMP-2 + 2.5, 5.0 or 10.0 ng/ml TNF-α for 3 days and ALP activity was assessed. **P<0.01 vs. BMP-2 group (0 ng/ml TNF-α). TNF, tumor necrosis factor; BMP, bone morphogenetic protein; ALP, alkaline phosphatase.

Article Snippet: In the present study, C2C12 cells were treated with BMP-2 (10426-H01H-10; Sinobiological, Inc., Beijing, China) in order to produce osteoblast cells.

Techniques: Activity Assay

Journal: Experimental and Therapeutic Medicine

Article Title: Triptolide inhibits the function of TNF-α in osteoblast differentiation by inhibiting the NF-κB signaling pathway

doi: 10.3892/etm.2017.4749

Figure Lengend Snippet: TPL significantly attenuated TNF-α-induced inhibition of osteoblast differentiation. C2C12 cells were treated with 100 ng/ml of BMP-2 and 5 ng/ml of TNF-α with or without triptolide (4, 8, 16 ng/ml) for 3 days and ALP activity was assessed. **P<0.01, vs. BMP-2-TNF-α group and #P<0.05, ##P<0.01, vs. BMP-2 group. TPL, triptolide; TNF, tumor necrosis factor; BMP, bone morphogenetic protein; ALP, alkaline phosphatase.

Article Snippet: In the present study, C2C12 cells were treated with BMP-2 (10426-H01H-10; Sinobiological, Inc., Beijing, China) in order to produce osteoblast cells.

Techniques: Inhibition, Activity Assay

Journal: Oncogenesis

Article Title: Neuron-specific enolase promotes stem cell-like characteristics of small-cell lung cancer by downregulating NBL1 and activating the BMP2/Smad/ID1 pathway.

doi: 10.1038/s41389-022-00396-5

Figure Lengend Snippet: Fig. 5 NSE downregulates and interacts with NBL1 in SCLC cells. A, B Western blot images showed the interaction between NSE and NBL1 by Co-IP experiment. C The double-immunofluorescence labeling experiment detected NSE and NBL1 and displayed co-location in clinical samples (original magnification ×40, scale bar 50 μm). D Western blot images show the interaction of BMP2 and BMPR1A enhanced after overexpression of NSE. E The photo shows a significant and negative correlation between ENO2 (encode NSE) and NBL1 in mRNA level using the CCLE database.

Article Snippet: The membranes were incubated with the following antibodies: NSE (1:2000, Abcam), GFP (1:2000, Proteintech), Flag (1:1000, Proteintech), OCT4 (1:1000, CST), Nanog (1:2000, CST), SOX2 (1:1000, CST), β-actin (1:5000, Proteintech), BMP2 (1:1000, Proteintech), Smad1 (1:2000, Proteintech), pSmad1/5/8 (1:1000, CST), and ID1 (1:500, Proteintech).

Techniques: Western Blot, Co-Immunoprecipitation Assay, Labeling, Over Expression

Journal: Immunity

Article Title: Stellate Cells, Hepatocytes, and Endothelial Cells Imprint the Kupffer Cell Identity on Monocytes Colonizing the Liver Macrophage Niche

doi: 10.1016/j.immuni.2019.08.017

Figure Lengend Snippet:

Article Snippet: Recombinant Human/Mouse/Rat BMP-2 Protein , RnD Systems , Cat#355-BM-010.

Techniques: Control, Recombinant, Blocking Assay, Irradiation, Enzyme-linked Immunosorbent Assay, Microarray, Software, Microscopy

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: An in vivo Comparison Study Between Strontium Nanoparticles and rhBMP2

doi: 10.3389/fbioe.2020.00499

Figure Lengend Snippet: Schematic representation of abiotic and in vivo sponge characterization. The collagen sponges were loaded with calcium hydroxyapatite nanoparticles (HAn), with calcium hydroxyapatite nanoparticles + BMP2 (HAn-BMP2), and a mixture of calcium hydroxyapatite nanoparticles and strontium hydroxyapatite nanoparticles (SrHAn). Sponge characterization was achieved by several physical-chemical techniques. X-ray observations and Fast green/Safranin-O histological staining were performed on tissue samples collected from the in vivo experiments. Expression of genes for osteogenesis, osteocytes and osteoclasts activity, chondrogenesis, and stem cell recruitment was also investigated. Figure created with BioRender.com .

Article Snippet: For rhBMP2 loaded-sponges, 3 μg of human recombinant BMP2 (R&D Systems) were added to the HAn suspension before the sponge absorption phase (HAn-BMP2).

Techniques: In Vivo, Staining, Expressing, Activity Assay

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: An in vivo Comparison Study Between Strontium Nanoparticles and rhBMP2

doi: 10.3389/fbioe.2020.00499

Figure Lengend Snippet: Calcium, strontium and rhBMP2 release from loaded untreated sponges. ICP-OES data representation of calcium and strontium ions release from HAn, HAn-BMP2, and SrHAn loaded sponges. Indirect ELISA was performed to quantify solubilized rhBMP2. Data were collected during the 28 days in aqueous solution. Ca 2+ released from HAn sponges (A) ; Ca 2+ and Sr 2+ released from SrHAn sponges (B) ; Ca 2+ and rhBMP2 release from HAn-BMP2 sponges (C,D) N = 3 samples were measure per each time point.

Article Snippet: For rhBMP2 loaded-sponges, 3 μg of human recombinant BMP2 (R&D Systems) were added to the HAn suspension before the sponge absorption phase (HAn-BMP2).

Techniques: Indirect ELISA

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: An in vivo Comparison Study Between Strontium Nanoparticles and rhBMP2

doi: 10.3389/fbioe.2020.00499

Figure Lengend Snippet: Tabular representation of the data shown in Figure 2.

Article Snippet: For rhBMP2 loaded-sponges, 3 μg of human recombinant BMP2 (R&D Systems) were added to the HAn suspension before the sponge absorption phase (HAn-BMP2).

Techniques:

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: An in vivo Comparison Study Between Strontium Nanoparticles and rhBMP2

doi: 10.3389/fbioe.2020.00499

Figure Lengend Snippet: The microscopic structure (150× and 350×) of the untreated CTRL (unloaded sponges), HAn, HAn-BMP2 and SrHAn (loaded sponges) samples is shown in (A-H) . Pictures represented samples at T0 (A–D) and after 28 days (E–H) of permanence in aqueous solutions, in a controlled and humidified atmosphere (37°C, with 5% of CO 2 ). Untreated samples surfaces were also analyzed with SEM-EDS (Energy Dispersive X-Ray Spectroscopy) at 0 (I,J) and 28 days (K,L) . Images at low magnifications of the sponges were taken with SEM (I–L) . The elements phosphorus (P), calcium (Ca) and strontium (Sr) were mapped on the same pictures using different colors (P = green; Ca = red; Sr = white) and a black background. Quantification of the elements carbon (C), oxygen (O), phosphorus (P), calcium (Ca) and strontium (Sr) present of the samples surface was performed and data were reported as weight percentage in the bar charts (M,N,O,P) histograms.

Article Snippet: For rhBMP2 loaded-sponges, 3 μg of human recombinant BMP2 (R&D Systems) were added to the HAn suspension before the sponge absorption phase (HAn-BMP2).

Techniques: Spectroscopy

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: An in vivo Comparison Study Between Strontium Nanoparticles and rhBMP2

doi: 10.3389/fbioe.2020.00499

Figure Lengend Snippet: X-ray images and macroscopic observation of mice implanted with loaded sponges. Images at 16 and 33 days of mice implanted with HAn loaded-sponges ( A,D,G,J,M,P , respectively), HAn-BMP2 loaded-sponges (B,E,H,K,N,Q) and SrHAn ( C,F,I, L,O,R , respectively). The implanted sponges are shown by black arrowheads. Isolated femurs are shown in panels (D–F) and (M–O) . Isolated legs including implants are shown in panels (G–I) and (P–R) .

Article Snippet: For rhBMP2 loaded-sponges, 3 μg of human recombinant BMP2 (R&D Systems) were added to the HAn suspension before the sponge absorption phase (HAn-BMP2).

Techniques: Isolation

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: An in vivo Comparison Study Between Strontium Nanoparticles and rhBMP2

doi: 10.3389/fbioe.2020.00499

Figure Lengend Snippet: Representative histological images of post-implants tissues of mouse limb. Fast green/Safranin-O staining was used on post-implant tissue sections of limbs implanted with loaded sponges with HAn (A,D,G,J) , HAn-BMP2 (B,E,H,K) , or SrHAn (C,F,I,L) , for 16 and 33 days, respectively. In the images, red spots of cartilaginous tissue are indicated with red arrowheads and gray blurs of ectopic bone are highlighted with *. Black arrows and black arrowheads indicate femur bone and sponges, respectively. The circle highlights a blood vessel.

Article Snippet: For rhBMP2 loaded-sponges, 3 μg of human recombinant BMP2 (R&D Systems) were added to the HAn suspension before the sponge absorption phase (HAn-BMP2).

Techniques: Staining

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: An in vivo Comparison Study Between Strontium Nanoparticles and rhBMP2

doi: 10.3389/fbioe.2020.00499

Figure Lengend Snippet: Gene expression of stem cell recruitment and chondrogenesis markers in bone and sponge post-implants. Gene expression was analyzed in femur bone (A,B,E,F) and sponge post-implant (C,D,G,H) samples at 16 (A–D) and 33 days (E–H) , respectively. Expression of chondrogenesis markers Acan, Col10A1 and Sox9 (A,C,E,G) as well as stem cell recruitment marker Nanog (B,D,F,H) has been evaluated. The graphs show the inverse of the ΔΔCt at the power of 2. Bars indicate mean values ± SEM of results from 4 experiments. Statistical significance values were calculated with one way-ANOVA, followed by Tukey's honestly significant difference test. *, significant difference against the HAn condition ( p < 0.05). #, significant difference between HAn-BMP2 and Sr conditions ( p < 0.05).

Article Snippet: For rhBMP2 loaded-sponges, 3 μg of human recombinant BMP2 (R&D Systems) were added to the HAn suspension before the sponge absorption phase (HAn-BMP2).

Techniques: Expressing, Marker

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: An in vivo Comparison Study Between Strontium Nanoparticles and rhBMP2

doi: 10.3389/fbioe.2020.00499

Figure Lengend Snippet: Gene expression of osteocytes, osteoclasts homeostasis, and osteogenic markers in bone and sponge post-implants. Gene expression was evaluated in femur bone (A,B,C,G,H,I) and sponge post-implant (D,E,F,J,K,L) samples at 16 (A–F) and 33 (G–L) days, respectively. Expression of osteogenic markers Dmp1, Bglap, Ibsp, Sp7 and Runx2 (A,D,G,J) , osteocytes marker Sost (B,E,H,K) and osteoclasts relevant markers Acp5, Rankl and Ctsk (C,F,I,L) has been evaluated. The graphs show the inverse of the ΔΔCt at the power of 2. Bars indicate mean values ± SEM of results from four experiments. Statistical significance values were calculated with one way-ANOVA, followed by Tukey's honestly significant difference test. *, significant difference against the HAn condition ( p < 0.05). #, significant difference between HAn-BMP2 and SrHAn conditions ( p < 0.05).

Article Snippet: For rhBMP2 loaded-sponges, 3 μg of human recombinant BMP2 (R&D Systems) were added to the HAn suspension before the sponge absorption phase (HAn-BMP2).

Techniques: Expressing, Marker

Journal:

Article Title: PROTEIN KINASE A REGULATES GDNF/RET-DEPENDENT BUT NOT GDNF/RET-INDEPENDENT URETERIC BUD OUTGROWTH FROM THE WOLFFIAN DUCT

doi: 10.1016/j.ydbio.2010.08.029

Figure Lengend Snippet: Wolffian ducts were isolated from E13 rat embryos and cultured for two days in either control media (FGF1 and GDNF) or in the added presence 5nM BMP. A) BMP2 completely inhibited UB outgrowth from the Wolffian duct. Green – E-cadherin; blue – DAPI. Scale bar = 50µm. B) PKA enzyme activity, normalized by protein concentration assay, was calculated from the slope of the linear least-squares fit of the PKA assay in each condition; the increase in PKA enzyme activity measured in those ducts exposed to BMP2 was determined to be statistically significant C) qRT-PCR confirms significant downregulation of Ret expression in the WDPKA+ although Ret levels in the WD exposed to BMP2 were not significantly different from control. D) qRT-PCR shows significant downregulation of GFRα expression in both the WDPKA+ and WDs cultured with 5nM BMP-2 in the absence of PKA inhibitor (* = p < 0.05 versus control; NS = not significant). Scale bar = 100µm.

Article Snippet: Recombinant GDNF, fibroblast growth factor (FGF)-1, FGF-7, follistatin, and bone morphogenetic protein (BMP)-2 were purchased from R&D systems (Minneapolis, MN).

Techniques: Isolation, Cell Culture, Activity Assay, Protein Concentration, Protein Kinase A Assay, Quantitative RT-PCR, Expressing