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. 2017 Aug 29;7:9628. doi: 10.1038/s41598-017-10182-8

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© The Author(s) 2017

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Incorporation of collagen networks in 3D hydrogel scaffolds supports cell alignment and differentiation. (a) Dry fabrics can be readily incorporated into commonly-used non-cell-adherent hydrogels such as agarose. (b) Low magnification epifluorescence images of actin-phalloidin and Hoechst nuclear staining demonstrate that collagen-doped fabrics promote large-scale alignment of agarose-encapsulated C2C12 cells. Cell alignment and growth is not observed for control fabrics that do not contain collagen. Arrows indicate direction of fabric alignment. (c) High-resolution confocal maximum intensity projections show details of aligned fascicle-like structures. Positioning of cell nuclei and interconnected cytoskeleton structures are indicative of myotube formation. Myotubes and fascicle-like structures are not observed for cells cultured with control fabrics. C2C12 cells growing along collagen networks within the hydrogels display immunofluorescence for skeletal muscle cell differentiation markers including myosin heavy chain, parvalbumin and NOS-I. All scale bars, 50 µm. (d) Representative Western blots for myosin heavy chain, parvalbumin and GAPDH for cells grown in 2D on tissue culture plastic, within agarose containing 3D control fabrics (3D (−)) and within agarose containing 3D collagen fabrics (3D (+)). Images are cropped from the original full blots with adjustment of brightness and contrast to ensure that faint bands are visible. Unprocessed Western blot images are available in Supplementary Figure 7. Levels of myosin heavy chain (e) and parvalbumin (f) relative to GAPDH (AU; Arbitrary Units) were compared across culture conditions. Significant differences of p < 0.05 by one-way ANOVA on ranks and Dunnett’s multiple comparison test with respect to the 2D control group are indicated by ^*. Densitometry data are available in Supplementary Table 1.