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Fabrication of RGD Micro/Nanopattern and Corresponding Study of Stem Cell Differentiation

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    Fabrication of RGD Micro/Nanopattern and Corresponding Study of Stem Cell Differentiation
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    State Key Laboratory of Molecular Engineering of Polymers, Department of Macromolecular Science, Advanced Materials Laboratory, Fudan University, Shanghai 200433, China
    *E-mail: [email protected]. Phone: 86 21 65643506. Fax: 86 21 65640293.
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    Nano Letters

    Cite this: Nano Lett. 2015, 15, 3, 1457–1467
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    https://doi.org/10.1021/nl5049862
    Published February 20, 2015
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    Abstract

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    Micropatterns of gold (Au) nanoarrays on inorganic and polymeric substrates were fabricated by combining block copolymer micelle nanolithography to obtain gold nanoarrays on glass, photolithography plus hydrofluoric acid (HF) etching to generate microislands, and transfer lithography to shift the gold micro/nanopatterns from glass to a bioinert poly(ethylene glycol) (PEG) hydrogel surface. Further the modification of the gold nanodots via cell-adhesive arginine-glycine-aspartate (RGD) ligands was carried out to achieve peptide micro/nanopatterns. Whereas the micro/nanopatterns of noble metals could be useful in various applications, the peptide micro/nanopatterns especially enable persistent cell localization on adhesive micropatterns of RGD nanoarrays on the background of potently nonfouling PEG hydrogels, and thus offer a powerful tool to investigate cell-material interactions on both molecular and cellular levels. As a demonstration, we cultured human mesenchymal stem cells (hMSCs) on micro/nanopatterns with RGD nanoarrays of nanospacings 46 and 95 nm, and with micropans of side lengths 35 and 65 μm (four groups in total). The osteogenic and adipogenic differentiation of hMSCs was conducted, and the potential effect of RGD nanospacing and the effect of cell spreading size on cell differentiation were decoupled for the first time. The results reveal that RGD nanospacing, independent of cell spreading size, acts as a strong regulator of cell tension and stem cell differentiation, which cannot be concluded unambiguously based on either merely micropatterns or nanopatterns.

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    FE-SEM images of the micro/nanopatterns of varied shapes of microdomains, global photograph of an as-prepared hydrogel with micro/nanopattern, AFM images of micropattern, AFM image of Au nanopattern on glass, micro/nanopattern at the boundary region of micropans on PEG hydrogels, schematic presentation of the reason that an overexposure would cause images of F-actins change color after recorded by a color CCD, distribution of spreading areas of free cells on nanopatterned surfaces, spreading areas of single hMSCs on micro/nanopatterned surfaces with different nanospacings and microisland sizes, representative histograms of pixel level distributions of F-actin-stained cell images, micrographs of osteogenesis of aggregate cells, relative integrated fluorescence intensity of fluorescence images of cells stained by p-MYL9, OPN and PPARγ, tables with fabrication parameters and corresponding characteristics of nanopatterns prepared in this study, and p-values in t-tests of the figures in the main manuscript and Supporting Information. This material is available free of charge via the Internet at http://pubs.acs.org.

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    Cite this: Nano Lett. 2015, 15, 3, 1457–1467
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    https://doi.org/10.1021/nl5049862
    Published February 20, 2015
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