71 research outputs found

    Chemotactic responses of MSCs.

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    <p>GFP-expressing MSCs (4×10<sup>4</sup>) were seeded onto the top of transwell chambers, with various cytokines/chemokines placed in the bottom of the chambers; some wells contained serum-free media (SFM) as a negative control. After a 20 hr incubation at 37°C, the GFP-MSCs that had migrated across the transwell membrane were lysed and quantitated by measuring fluorescence intensity of GFP. The following chemoattractants were evaluated: <b><i>A)</i></b> recombinant human PDGF-BB, PDGF-AB, or a mixture of SDF-1α, CXCL16, MIP-1α, MIP-1β, and RANTES, each at the indicated concentrations (ng/mL) (representative of 3 independent runs) <b><i>B)</i></b> PDGF-BB and BMP-2 (representative of 3 independent runs) and <b><i>C),</i></b> varying concentrations of PDGF-BB showing dose response.</p

    PDGF-BB released from PCL/col/HA scaffolds stimulates MSC chemotaxis.

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    <p>The lower wells of transwell chambers were filled with either purified PDGF-BB (10 ng/mL), serum-free medium (SFM) or PDGF-BB-containing conditioned media collected from PDGF-BB-coated PCL/col/HA scaffolds after 72 hrs. GFP-MSCs were seeded in the upper chambers and allowed to migrate for 20 hrs. After this interval, MSCs adherent to the underside of the transwells were visualized by fluorescent microscopy (top panel, representative images). In addition, MSC migration to the underside of the filter was quantified by lysing cells and measuring solution fluorescence (lower panel). Three independent experiments were performed for solution fluorescence. Analysis of variance with Tukey’s HSD post-hoc was used to establish significance (* denotes <i>p</i><.01).</p

    A patient with a 0.5-year history of pseudophakic bullous keratopathy.

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    <p>(A) A slit-lamp photograph of the cornea showing no obvious scar formation; (B) in the histological sections, no obvious scars, neovascularization or inflammatory cells can be observed; (C) Masson's trichrome staining; and (D) Van Gieson staining. <b>Scale bar 50 µm.</b></p

    Standard curve of GFP fluorescent signal from lysed GFP-MSCs.

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    <p>GFP-MSCs were counted using a hemocytometer and set numbers of cells were spun down in a centrifuge. Cell pellets were lysed and solution fluorescence was measured by a fluorometer. The coefficient of determination for the linear regression was 0.999, showing a very strong linear correlation between GFP-MSC number and solution fluorescence.</p

    Released PDGF-BB induces chemotaxis of MSCs in a stringent migration assay. <i>A)</i>

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    <p>Schematic showing experimental set-up (not drawn to scale). GFP-MSCs were seeded in 8-well rectangular plates. After cell confluence was established, cells were completely removed from the top half of the well by scraping along a pre-drawn central line. Subsequently, a PDGF-BB-adsorbed PCL/col/HA/scaffold, placed on a steel wire mesh, was placed 1.5 cm away from the cell front. As a control, some chambers were set up with PCL/col/HA scaffolds lacking PDGF-BB. <b><i>B)</i></b> After a 72 hr-incubation in the chambers described, MSCs were stained with DAPI and visualized by fluorescence microscopy. The original cell front created is denoted by a white line. <b><i>C)</i></b> GFP-images showing change in cell morphology of MSCs exposed to PDGF-BB. <b><i>D)</i></b> Significantly greater cell number was observed migrating toward PDGF-BB coated scaffolds compared to uncoated scaffolds. <b><i>E)</i></b> DAPI-stained images were further analyzed by counting the number of cells in three defined regions of distance beyond the original cell front. The distribution of cells in the wells with PDGF-BB-coated scaffolds showed that a greater percentage of the total cells that had migrated beyond the cell front had localized to the region beyond 400 µm. In comparison, the greatest percentage of cells in the control wells localized to the region below 150 µm. A total of six samples were analyzed for each condition. An asterisk (*) denotes significant differences observed with <i>p</i><.01, whereas (**) denotes <i>p</i><.0001.</p

    Adsorption and release of PDGF-BB from scaffolds. <i>A)</i>

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    <p>Scaffolds were incubated in PBS containing 1.5 µg PDGF-BB for 24 h at 4°C. ELISA assays were used to measure the unbound PDGF-BB in the supernatants. Adsorption of PDGF-BB to the scaffolds was determined by subtracting the unbound PDGF-BB from the 1.5 µg of PDGF-BB initially added. Data are from three independent experiments (* denotes p<0.01). <b><i>B)</i></b> ELISAs were used to measure the amounts of PDGF-BB in conditioned PBS solution collected from the scaffolds at the indicated time intervals over a period of 8 weeks (for many of the data points, error bars are too small to be visualized).</p

    Aqueous Polymerization-Induced Self-Assembly for the Synthesis of Ketone-Functionalized Nano-Objects with Low Polydispersity

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    Efficient synthesis of functionalized, uniform polymer nano-objects in water with controlled morphologies in one step and at high concentrations is extremely attractive, from perspectives of both materials applications and industrial scale-up. Herein, we report a novel formulation for aqueous reversible addition–fragmentation chain transfer (RAFT) dispersion polymerization based on polymerization-induced self-assembly (PISA) to synthesize ketone-functionalized nanospheres and vesicles. Significantly, the core-forming block was composed entirely of a ketone-containing polymer from a commodity monomer diacetone acrylamide (DAAM), resulting in a high density of ketone functionality in the nano-objects. Producing uniform vesicles represents another challenge both in PISA and in the traditional self-assembly process. Aiming at producing uniform nano-objects, especially vesicles, in such a highly efficient aqueous PISA process, we devised strategies to allow sufficient time for the in situ generated polymers to relax and reorganize into vesicles with a remarkably low polydispersity. Specifically, both reducing the radical initiator concentration and lowering the polymerization temperature were shown to be effective for improving the uniformity of vesicles. Such an efficient, aqueous PISA to produce functionalized and uniform nano-objects with controlled morphologies at solid contents up to 20% represents important progress in the field

    Exon structure of cardiac muscle expressed nNOS splice variants and their expression profile in the nNOS null mouse models used in this study.

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    <p>Coding exons of each splice variant are gray numbered boxes. Exon 2 encodes the PDZ (PSD95/Dlg1/ZO1) protein-protein interaction domain. Exon 6 encodes the heme binding domain essential for nitric oxide synthesis. Exon sequences that form unique 5’ untranslated sequences are white. Asterisks mark translation initiation sites. nNOS splice variant expression in control and murine KN1 and KN2 nNOS knockout models are shown on the right. A tick mark indicates expression. A cross mark indicates absence of expression and/or activity. This study employs two nNOS knockout models: first knockout of nNOS (KN1, exon 2 deletion) and second knockout of nNOS (KN2, exon 6 deletion). KN1 and KN2 mice have distinct isoform expression profiles. KN1 mice lack nNOSα and nNOSμ, but still express nNOSβ. KN2 mice do not express any active nNOS splice variants.</p

    Evaluation of morphological changes in patients with bullous keratopathy.

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    <p>Significant differences were found between the more than 1.0 year group and the less than 1.0 year group (*, P<0.05), including (A) corneal stromal scar grading, (B) the number of new vessels per high-power field, and (C) the number of inflammatory cells per high-power field.</p

    CTGF and TGF-β expression in the more than 1.0 year group.

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    <p>(A) The CTGF expression in the deep stroma (arrow); and (B) the TGF-β expression in the deep stroma (arrow). <b>Scale bar 20 µm.</b></p
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