Journal: bioRxiv

Article Title: Capturing trophectoderm-like stem cells enables step-wisely remodeling of placental development

doi: 10.1101/2025.08.25.672082

Figure Lengend Snippet: (A) The morphology of ESCs, TBLCs and ESCs, TBLCs in TS medium after 3 days of induction. Scale bars, 250 μm. (B) FACS analysis of the percentage of CDX2 + cells from ESCs and TBLCs, as well as ESCs and TBLCs cultured in TS medium, using V6.5 cell line. (C) FACS analysis of the percentage of CD40 + cells from ESCs and TBLCs, as well as ESCs and TBLCs cultured in TS medium, using TC1 cell line. (D) FACS analysis of the percentage of CD40 + TELCs obtained from the TBLCs after induction with different molecules, including FGF4, Activin A, TGFβ1 and BMP4. The corresponding cell morphology is displayed in the lower panel. (E) Scatterplots displaying the transcriptome comparison of TELCs before and after CD40-based FACS using RNA-seq. Upregulated (FC>2) and downregulated (FC<0.5) genes are shown in red and blue, respectively. (F) The morphology of TBLCs of different passages and long-term culture in TX and TS medium, also the morphology of TBLCs after CD40 FACS after induction. Scale bars, 250 μm. (G) Western blotting was used to detect OCT4, CDX2 and EOMES in TELSCs from different passages. β-Tubulin was used as a loading control. (H) The morphology 8C embryos cultured in TX medium. Scale bars, 250 μm. (I) FACS analysis of the percentage of CD40 + cells in TELSC em s at different passages. (J) Immunofluorescence staining of TFAP2C and PEG10 in TBLCs, TELSCs and TELSC em s. Scale bars, 50 μm. (K) Cell cycle analysis of ESCs, TELSCs and TELSC em s. (L) Heatmap indicating the relative expression of TBLCs, TELSCs and TELSC em s. The representative genes and enrichment of GO terms of these genes is shown. (M) Heatmap indicating the relative expression of characteristic genes in TELSCs, TELSC em s and TSCs. Bubble chart showing the relative expression of these genes in mouse embryos. (N) Heatmap indicating the relative expression of characteristic genes in TELSCs, TSCs cultured in TX medium and TSCs cultured in TS medium. Heatmap on the right demonstrating the expression of each cluster in mouse embryos. The representative genes and enrichment of GO terms of these genes is shown. (O) The scatter plot displays differentially expressed genes between TELSCs and TSCs cultured in various media. The bar graph summarizes the number of differentially expressed genes identified under each comparison condition. (P) GSEA analysis of ESCs, TBLCs, TELCs and TELSCs based on “embryonic placenta development” and “placenta development” geneset. (Q) Heatmap indicating the differentially expressed genes in Hippo pathway of TELSCs and TBLCs. (R) Heatmap indicating the relative expression of characteristic genes in TELSCs, TSCs cultured in TX medium and TSCs cultured in TS medium. Bubble chart showing the relative expression of these genes in mouse embryos. (S) Phase contrast images of TBLCs cultured in TS medium for 24h supplemented with Verteporfin at the indicated concentration. Scale bars, 100 µm. (T) Heatmap indicating the differentially expressed genes of TELCs and TBLCs induction in TS medium plus verteporfin. Bubble chart showing the relative expression of these genes in mouse embryos. (U) GSEA analysis of TELCs, TBLCs induction in TS medium and in TS medium plus verteporfin based on TE geneset. (V) The morphology of TELSCs cultured in TS medium, TS medium plus ITS-X and TS medium plus TGFβ1. (W) Heatmap indicating the differentially expressed genes of TELSCs, TBLCs induction in TX medium withdraw ITS-X, in TS medium and in TS medium plus ITS-X. Heatmap on the right demonstrating the expression of each cluster in mouse embryos. The representative genes and enrichment of GO terms of these genes is shown. (X) GSEA analysis of TBLCs induction in TX medium withdraw ITS-X and in TX medium based on “Positive regulation of stem cell proliferation” and “Positive regulation of cell cycle” geneset.

Article Snippet: All TSLs were cultured on Matrigel-coated plates, in 30% TS medium (RPMI 1640 (GIBCO, 11875119), 20% FBS, 1% GlutaMax (GIBCO, 35050061), 1% penicillin-streptomycin (GIBCO, 15140163), 1% sodium pyruvate (GIBCO, 11360070)) and 70% MEF-conditioned TS medium supplemented with 25 ng/ml human recombinant FGF4 (MCE, HY-P7014) and 1 μg/ml heparin (STEMCELL, 7980).

Techniques: Cell Culture, Comparison, RNA Sequencing, Western Blot, Control, Immunofluorescence, Staining, Cell Cycle Assay, Expressing, Concentration Assay

Journal: bioRxiv

Article Title: Ubiquitin Ligase ITCH Regulates Life Cycle of SARS-CoV-2 Virus

doi: 10.1101/2024.12.04.624804

Figure Lengend Snippet: (A) Culture medium from vT2-WT and vT2-ITCH-KO cells expressing HA-tagged ubiquitin (Ub) and Flag-2×Strep tagged E was harvested for Strep AP. A decrease of the extracellular E protein induced by ITCH ablation was visualized by immunoblot (left) and dot blot analysis (right). (B) Lysates from HEK293 cells expressing Flag-tagged E with ITCH or ITCH-CS were subjected to Flag IP, followed by immunoblotting for autophagosome cargo receptors. E specifically precipitated p62 and ITCH promoted their interaction. (C-E) HEK293 cells were transfected with Flag-tagged E with ITCH or ITCH-CS. 24 h later, cells were analyzed by immunofluorescence with Flag, ITCH and p62 or LC3B or LAMP1 antibodies. ITCH enhanced the colocalization between E and p62 (C) or LC3B (D), while no change in the colocalization between E and LAMP1 (E) was noted. Scale bar, 10 μm. (F) Culture media and denatured lysates from control (CTRL) and p62 knock down (#1, #2) HEK293 cells expressing HA-tagged ubiquitin (Ub), Flag-tagged E were subjected to Flag IP (with incorporation of a washing step with urea before elution for culture media samples), followed by immunoblotting or dot blot analysis. p62 depletion resulted in the accumulation of intracellular E (both unmodified and ubiquitinated), while decreasing the level of extracellular E (n=3). (G, H) vT2-WT and vT2-ITCH-KO cells infected with SARS-CoV-2 at 1 MOI for 10 h were subjected to immunofluorescence analysis with E and p62 or LC3B antibodies. ITCH-ablation decreased the colocalization between E and p62 (G) or LC3B (H). Scale bar, 10 μm. (I) A model of the function of ITCH in promoting autophagosome-mediated SARS-CoV-2 virion egress. ITCH-dependent ubiquitin modification enhances E binding with S and M binding with non-ubiquitinated E, resulting in the increase in virion formation and p62-dependent autophagosome targeting for release.

Article Snippet: The following primary antibodies were used: Flag (Sigma, F1804); Flag (Sigma, F3165); Flag (Cell Signaling Technology, 14793S); GAPDH (Invitrogen, MA5-27912); β-tubulin (Cell Signaling Technology, 2128S);s (BioLegend, 688102); Strep (Invitrogen, MA5-17283); CBD (New England BioLabs, E8034S); ubiquitin (Cell Signaling Technology, 58395S); K63-linkage-specific antibody (Enzo Life Sciences, BML-PW0600-0100); K48-linkage-specific antibody (Cell Signaling Technology, 8081S); Spike (Proteintech, 28867-1-AP); M (Proteintech, 28882-1-AP); E (Proteintech, 28904-1-AP); ITCH (Santa Cruz, sc-28367); ITCH (Novus Biologicals, NB100-68142); p62 (Cell Signaling Technology, 88588S and 7695S); GM130 (Proteintech, 11308-1-AP); LAMP1 (Cell Signaling Technology, 9091S); OPTN (Cayman Chemical, 100002); LC3 (Proteintech,14600-1-AP); LC3B (Cell Signaling Technology, 3868S); SARS-CoV-2 Membrane protein (Cell Signaling Technology, 15333S); SARS-CoV-2 Envelope protein (Cell Signaling Technology, 74698S); furin (Proteintech, 18413-1-AP); Cathepsin L (Proteintech, 10938-1-AP); NDP52 (Proteintech, 12229-1-AP); NBR1 (Proteintech, 16004-1-AP); FAM134B (Proteintech, 21537-1-AP); RTN3 (Proteintech, 12055-2-AP) and NIX (Proteintech, 12986-1-AP).

Techniques: Expressing, Western Blot, Dot Blot, Transfection, Immunofluorescence, Control, Knockdown, Infection, Modification, Binding Assay

Journal: bioRxiv

Article Title: RNA sequencing reveals key factors modulating TNFα-stimulated odontoblast-like differentiation of dental pulp stem cells

doi: 10.1101/2025.01.09.632294

Figure Lengend Snippet: (A ) Heatmap of the differentially expressed genes between untreated or control cells and TNFα hDPSCs in dentinogenic media. Red and yellow stripes in the figure represent high-expression genes, while blue stripes represent low-expression genes. (B) Volcano map of differentially expressed genes (DEGs) between control cells and TNFα hDPSCs in dentinogenic media. The x-axis is the log2 scale of the fold change of gene expression in hDPSCs (log2(fold change)). Negative values indicate downregulation; positive values indicate upregulation. The y-axis is the minus log10 scale of the adjusted p values (–log10), which indicate the significant level of expression difference. The blue dots represent significantly upregulated genes with at least twofold change, while the red dots represent significantly downregulated genes with at least twofold change. (C) Significant enriched Gene Ontology (GO) terms among control and TNFα treated hDPSCs based on their functions. The top GO terms in the enrichment analysis among biological process, cellular component and molecular function (MF) terms in the enrichment analysis.

Article Snippet: Human recombinant TNFα was from Invitrogen, Fisher Scientific (Waltham, MA, USA), and a few other chemicals were from Fisher Chemical (Nazareth, PA, USA). siRNA targeting human C5L2, siRNA control and siRNA Reagent System were purchased from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Control, Expressing

Journal: bioRxiv

Article Title: RNA sequencing reveals key factors modulating TNFα-stimulated odontoblast-like differentiation of dental pulp stem cells

doi: 10.1101/2025.01.09.632294

Figure Lengend Snippet: Go-term and Reactome enrichment pathway analysis of up- and down-regulated DEGs. Dot plot shows top enriched Reactome pathways. The size of the dot is based on gene count enriched in the pathway, and the color of the dot shows the pathway enrichment significance. (C-D) Gene set enrichment analysis was performed on DEGs among control and TNFα treated cells in dentinogenic media and found various upregulated and downregulated genes against inflammatory response (C) extracellular matrix structural constituents (D). (E) Sashimi plots for quantitative visualization of RNA sequencing read alignments. Data were examined on sashimi plots where it revealed the number of variants and genomic mutation on chr14 in TNFα treated cells in dentinogenic media against control. Red sashimi plots showing variants in TNFα treated group and orange shows in control. While lower black annotations are Read alignments of alternative isoforms and genomic region of interest.

Article Snippet: Human recombinant TNFα was from Invitrogen, Fisher Scientific (Waltham, MA, USA), and a few other chemicals were from Fisher Chemical (Nazareth, PA, USA). siRNA targeting human C5L2, siRNA control and siRNA Reagent System were purchased from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Control, RNA Sequencing Assay, Mutagenesis

Journal: bioRxiv

Article Title: RNA sequencing reveals key factors modulating TNFα-stimulated odontoblast-like differentiation of dental pulp stem cells

doi: 10.1101/2025.01.09.632294

Figure Lengend Snippet: hDPSCs were cultured and treated with or without TNFα for 7 days (twice a week with 3 days interval). Cell lysates were collected, and RNA were prepared using RNeasy mini kit (Qiagen), and next-generation RNA sequencing was done using poly-A-RNA sequencing technique. (A) Histogram showing upregulated and activated transcription factors (blue) and repressed or down-regulated transcription factors (orange). It is noteworthy that TCF (7, 12, 19, and 20) especially TCF12 highly up-regulated in TNFα-stimulated odontoblasts like differentiated DPSCs. (B) Histogram showing upregulated and activated up-regulated genes (blue) and repressed or down-regulated genes (orange). It is noteworthy that key genes that are involved in dentinogenesis are significantly up-regulated in TNFα-stimulated odontoblasts like differentiated DPSCs.

Article Snippet: Human recombinant TNFα was from Invitrogen, Fisher Scientific (Waltham, MA, USA), and a few other chemicals were from Fisher Chemical (Nazareth, PA, USA). siRNA targeting human C5L2, siRNA control and siRNA Reagent System were purchased from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Cell Culture, RNA Sequencing Assay

Journal: bioRxiv

Article Title: RNA sequencing reveals key factors modulating TNFα-stimulated odontoblast-like differentiation of dental pulp stem cells

doi: 10.1101/2025.01.09.632294

Figure Lengend Snippet: Significant enriched GO terms can be found in TNFα treated hDPSCs in dentinogenic media based on their functions compared with control. (A-B) Go-term and Reactome enrichment pathway analysis of up- and down-regulated DEGs. Dot plot shows top enriched Reactome pathways. The size of the dot is based on gene count enriched in the pathway, and the color of the dot shows the pathway enrichment significance. (C) Gene set enrichment analysis was performed on DEGs among control and siC5L2 treated cells in dentinogenic media and found various upregulated and downregulated genes against bacterial response. Enrichment plot and Random ES distribution shows defense response against bacterium have been enhanced after C5L2 silencing. (D) Sashimi plots for quantitative visualization of RNA sequencing read alignments. Data were examined on sashimi plots where it revealed the number of variants and genomic mutation on chr16, chr19 and chr11 in siC5L2 treated cells in dentinogenic media against control. Red sashimi plots showing variants in siC5L2 treated group and orange shows in control. While lower black annotations are Read alignments of alternative isoforms and genomic region of interest.

Article Snippet: Human recombinant TNFα was from Invitrogen, Fisher Scientific (Waltham, MA, USA), and a few other chemicals were from Fisher Chemical (Nazareth, PA, USA). siRNA targeting human C5L2, siRNA control and siRNA Reagent System were purchased from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Control, RNA Sequencing Assay, Mutagenesis

Journal: bioRxiv

Article Title: RNA sequencing reveals key factors modulating TNFα-stimulated odontoblast-like differentiation of dental pulp stem cells

doi: 10.1101/2025.01.09.632294

Figure Lengend Snippet: (A ) Heatmap of the differentially expressed genes between untreated or control cells and TNFα treated C5L2 silenced hDPSCs in dentinogenic media. Red and yellow stripes in the figure represent high expression genes, while blue stripes represent low expression genes. (B) Volcano map of differentially expressed genes (DEGs) between control cells and siC5L2 hDPSCs in dentinogenic media. The x-axis is the log2 scale of the fold change of gene expression in hDPSCs (log2(fold change)). Negative values indicate downregulation; positive values indicate upregulation. The y-axis is the minus log10 scale of the adjusted p values (–log10), which indicate the significant level of expression difference. The blue dots represent significantly upregulated genes with at least twofold change, while the red dots represent significantly downregulated genes with at least twofold change. (C) Significant enriched Gene Ontology (GO) terms among control and TNFα+siC5L2 treated hDPSCs based on their functions.

Article Snippet: Human recombinant TNFα was from Invitrogen, Fisher Scientific (Waltham, MA, USA), and a few other chemicals were from Fisher Chemical (Nazareth, PA, USA). siRNA targeting human C5L2, siRNA control and siRNA Reagent System were purchased from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Control, Expressing

Journal: bioRxiv

Article Title: RNA sequencing reveals key factors modulating TNFα-stimulated odontoblast-like differentiation of dental pulp stem cells

doi: 10.1101/2025.01.09.632294

Figure Lengend Snippet: Significant enriched GO terms can be found in TNFα treated C5L2 silenced hDPSCs in dentinogenic media based on their functions compared with control. (A-B) Go-term and Reactome enrichment pathway analysis of up- and down-regulated DEGs. Dot plot shows top enriched Reactome pathways. The size of the dot is based on gene count enriched in the pathway, and the color of the dot shows the pathway enrichment significance. (C) Sashimi plots for quantitative visualization of RNA sequencing read alignments. Data were examined on sashimi plots where it revealed the number of variants and genomic mutation on chr3, chr11, Chr10 and chr19 in TNFα treated C5L2 silenced DPSCs in dentinogenic media against control. Red sashimi plots showing variants in TNFα+siC5L2 treated group and orange shows in control. While lower black annotations are Read alignments of alternative isoforms and genomic region of interest.

Article Snippet: Human recombinant TNFα was from Invitrogen, Fisher Scientific (Waltham, MA, USA), and a few other chemicals were from Fisher Chemical (Nazareth, PA, USA). siRNA targeting human C5L2, siRNA control and siRNA Reagent System were purchased from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Control, RNA Sequencing Assay, Mutagenesis

Journal: bioRxiv

Article Title: RNA sequencing reveals key factors modulating TNFα-stimulated odontoblast-like differentiation of dental pulp stem cells

doi: 10.1101/2025.01.09.632294

Figure Lengend Snippet: Signaling pathways affected by C5L2 silencing in odontoblasts like differentiation of hDPSCs in dentinogenic media stimulated by TNFα . hDPSCs were cultured and treated with or without TNFα (twice a week) in C5L2 silenced DPSCs and differentiated for 7 days. Cell lysates were collected, and RNA were prepared using RNeasy mini kit (Qiagen), and next-generation RNA sequencing was done using poly-A-RNA sequencing technique. Histogram showing upregulated and activated transcription factors (blue) and repressed or down-regulated transcription factors (orange). It is noteworthy that TCF (4, 12, 20, and 25) especially TCF4 highly up-regulated in C5L2 silenced TNFα-stimulated odontoblasts like differentiated DPSCs.

Article Snippet: Human recombinant TNFα was from Invitrogen, Fisher Scientific (Waltham, MA, USA), and a few other chemicals were from Fisher Chemical (Nazareth, PA, USA). siRNA targeting human C5L2, siRNA control and siRNA Reagent System were purchased from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Cell Culture, RNA Sequencing Assay

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