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