Journal: Scientific Reports

Article Title: Nanogel tectonic porous 3D scaffold for direct reprogramming fibroblasts into osteoblasts and bone regeneration

doi: 10.1038/s41598-018-33892-z

Figure Lengend Snippet: Osteoblasts efficiently adhered to and proliferated on the fibronectin-coated NanoCliP-FD gel. ( a ) Preparation of NanoCliP-FD gel with dOBs. ( b , c ) XOL-transduced cells were seeded into rhodamine-labeled NanoCliP-FD gel that had been coated with either RGDC or fibronectin. After culturing for the indicated days, the samples were stained with calcein-AM ( b ) or phalloidin/Hoechst 33342 ( c ), and CLSM images at magnifications of objective lenses of x2.5 (upper) and x20 (lower) are shown. ( d ) XOL-transduced cells were seeded into non-coated (−), RGDC-conjugated, and fibronectin-coated NanoCliP-FD gel. After culturing for the indicated days, cell viability was evaluated by tetrazolium-based assay. Values are means ± SD. N = 3. **p < 0.01 vs. day 1.

Article Snippet: For fibronectin-coating, NanoCliP-FD matrix was soaked in 50 μg/mL fibronectin solution (Wako laboratory chemicals, Osaka, Japan) for 6 hours, followed by rinsing twice in ethanol and drying (Fig. ).

Techniques: Labeling, Staining

Journal: Scientific Reports

Article Title: Nanogel tectonic porous 3D scaffold for direct reprogramming fibroblasts into osteoblasts and bone regeneration

doi: 10.1038/s41598-018-33892-z

Figure Lengend Snippet: XOL-transduced cells were successfully converted into dOBs that produced calcified bone matrix in fibronectin-coated NanoCliP-FD gel. ( a ) RNA was extracted from the HDFs or XOL-transduced cells cultured in fibronectin-coated NanoCliP-FD gel for 14 and 28 days. mRNA levels for the indicated genes were evaluated by real time-RT-PCR. Values are means ± SD. N = 3. **p < 0.01 vs. HDFs. ( b , c ) Fibronectin-coated NanoCliP-FD gel with HDFs and XOL-transduced cells were cultured for the indicated days and stained with Alizarin red S. Some aliquots of the fibronectin-coated NanoCliP-FD gel were cultured without cell seeding (−). Macroscopic images ( b ) and relative staining intensities ( c ) are shown. Values are means ± SD. N = 3. **p < 0.01 vs. cell-free control. ( d ) Fibronectin-coated NanoCliP-FD gel with HDFs or XOL-transduced cells was cultured as above, and osteoimage assay was performed 21 days later. Confocal LSM images at low (upper) and high (lower) magnifications are shown.

Article Snippet: For fibronectin-coating, NanoCliP-FD matrix was soaked in 50 μg/mL fibronectin solution (Wako laboratory chemicals, Osaka, Japan) for 6 hours, followed by rinsing twice in ethanol and drying (Fig. ).

Techniques: Produced, Cell Culture, Quantitative RT-PCR, Staining

Journal: Scientific Reports

Article Title: Nanogel tectonic porous 3D scaffold for direct reprogramming fibroblasts into osteoblasts and bone regeneration

doi: 10.1038/s41598-018-33892-z

Figure Lengend Snippet: Bone healing was facilitated by transplantation of the fibronectin-coated NanoCliP-FD gel with dOBs. Fibronectin-coated NanoCliP-FD gel with HDFs or dOBs was prepared as in Fig. , and transplanted into an artificial segmental bone defect lesion that was created at femoral diaphysis in NOG/SCID mice. Control mice were not transplanted (−). Mice were sacrificed 21 days after the surgery. ( a , b ) µCT images of the femur were acquired. Serial 10-µm slices (top and middle) and 3D reconstructed (bottom) images ( a ) and %Callus formation ( b ) are shown. ( c ) Serial sections of the tissues were stained with H-E (upper) and Alizarin red S (lower). In ( a ) triangles and arrows represent bone defect lesions and regenerated bone tissue, respectively. In ( b ), values are means ± SD. N = 3 mice. **p < 0.01 vs. non-transplantation control. In ( c ), *and +represent regenerated bone tissue and NanoCliP-FD gel, respectively, and arrowheads represent bone defect lesions.

Article Snippet: For fibronectin-coating, NanoCliP-FD matrix was soaked in 50 μg/mL fibronectin solution (Wako laboratory chemicals, Osaka, Japan) for 6 hours, followed by rinsing twice in ethanol and drying (Fig. ).

Techniques: Transplantation Assay, Staining

Journal: Annals of Clinical and Translational Neurology

Article Title: Engineered extracellular matrices facilitate brain organoids from human pluripotent stem cells

doi: 10.1002/acn3.51820

Figure Lengend Snippet: Engineered Extracellular Matrix fabrication and neural differentiation. (A) Fabrication of Engineered Extracellular Matrices (EECMs) used for seeding ESCs and downstream organoid generation. 3D jet writing is used to produce poly( d , l ‐lactide‐ co ‐glycolide, PLGA) scaffolds, as previously described. ⁷² Polymer PLGA scaffolds are mounted onto medical‐grade stainless steel and then this structure is placed into a fibronectin solution that undergoes rotation to induce fibrillogenesis and deposit an insoluble fibronectin (FN) matrix. ²⁰ , ²² Human ESCs are then seeded onto EECMs. (B) Scanning electron micrograph (SEM) of the PLGA scaffold structure (scale bar is 200 μm). (C) Deposited fibronectin matrix is stained using an amine‐reactive fluorophore (red channel; scale bar is 500 μm). (D) SEM of confluent embryonic stem cells (ESCs; H9) cultured on the EECM (scale bar is 100 μm). (E) Timeline for brain organoid production using a commercial media kit. (F) A confluent stem cell layer is generated prior to Neural Induction Medium (NIM), stained for pluripotency markers SOX2 (green channel), OCT4 (red channel), and NANOG (yellow channel) plus merged image (top; scale bar is 30 μm). (G) EECMs with differentiated H9 ESCs after NIM, stained for ZO‐1 (pink channel; scale bar is 200 μm), SOX1 (yellow channel), and beta‐catenin (red channel) markers (scale bar in insets is 100 μm), identifying neural progenitor cells and neural rosette formation. (H) Neuronal populations stained for neuronal markers TUJ1 (green channel), OTX2 (purple channel), and synaptophysin (red channel), plus merged image, at the end of maturation (scale bar is 20 μm). Panels F–H are all counterstained with DAPI (cyan channel). (I) qPCR results showing the fold‐change of expression for neuronal differentiation markers of EECM organoids at D45 of culture relative to day 0 (undifferentiated cells); n ≥ 3 biological replicates, that is, three distinct batches of brain organoids, for each gene of interest. All genes are normalized to GAPDH expression levels. (J) Western blot showing protein expression for hPSCs before differentiation (D0) and EECM organoids at D45 of culture.

Article Snippet: To create EECMs of fibrillar fibronectin, this scaffolding was placed in a plasma fibronectin solution (Corning, Glendale, AZ) diluted in DPBS with no magnesium or calcium (Gibco, Grand Island, NY) to a concentration of 0.111 mg/mL and then tumbled in a microcentrifuge tube at 8 RPM in a 30°C chamber for at least 2 h. The air‐polymer‐protein interface induces fibrillogenesis of the fibronectin to convert the soluble protein to insoluble protein fibrils., The resulting structure is an ultraporous polymer scaffold laden with a fibrillar fibronectin protein matrix, referred to collectively as EECM.

Techniques: Polymer, Staining, Cell Culture, Generated, Expressing, Western Blot

Journal: Integrative biology : quantitative biosciences from nano to macro

Article Title: Regulation of epithelial cell organization by tuning cell-substrate adhesion

doi: 10.1039/c5ib00196j

Figure Lengend Snippet: Variations of ECM or cell-cell junction proteins changes the phenotypes of the colony. A. Representative images of cell colonies grown for 5 days. Colonies with different modifications of the CCJ apparatus were grown on 5 (top row) or 80 μg/ml (bottom) of either fibronectin or collagen coated substrates. Specific conditions for each column are as indicated. Standard conditions (wild type E-Cadherin based junction on FN substrate) are taken from Fig. 2. Day 5 was chosen as end point condition. B and C. Plots of colonies area and roundness after 5 days of expansion. Data in B and C are significantly different in a two-ways ANOVA analysis (p < 0.05). (Ecad/Cad11-Fibronectin = 6 and 6 independent colonies for both protein concentration; E-Cadherin-Collagen = 8 and 6 ind. col. for 5 and 80 μg/ml, respectively; E-Cadherin-Fibronectin = 7 and 3 ind. col. for 5 and 80 μg/ml, respectively; a-catenin KD-Fibronectin = 8 and 11 ind. col. for 5 and 80 μg/ml, respectively Error bars = standard error of the mean. D. Frequency (%) of angles of velocity vectors as compared to tissue center of mass. 90° is radial, 0° is orthoradial. Average of 2 independent colonies for each condition is shown.

Article Snippet: Preparation of cell seeding onto varying fibronectin concentration 600μl of varying concentration (5, 80μg/ml) of fibronectin solution was incubated for 1h at 37° C onto glass-bottom petri dish (IBIDI, Sciencewerke Pte Ltd.).

Techniques: Protein Concentration