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ATunable Scaffold of Microtubular Graphite for 3D Cell Growth

View Article: PubMed Central - PubMed

ABSTRACT

Aerographite(AG) is a novel carbon-based material that exists as a self-supportive3D network of interconnected hollow microtubules. It can be synthesizedin a variety of architectures tailored by the growth conditions. Thisflexibility in creating structures presents interesting bioengineeringpossibilities such as the generation of an artificial extracellularmatrix. Here we have explored the feasibility and potential of AGas a scaffold for 3D cell growth employing cyclic RGD (cRGD) peptidescoupled to poly(ethylene glycol) (PEG) conjugated phospholipids forsurface functionalization to promote specific adhesion of fibroblastcells. Successful growth and invasion of the bulk material was followedover a period of 4 days.

No MeSH data available.


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(A)YFP fluorescence (green) images of REF YFP-paxillin cells on a flatsubstrate, which have grown to a confluent layer. Bright fluorescentspots indicate focal adhesions of the cells. Intracellular homogeneousfluorescence originates in part from cytosolic paxillin. Dark circularregions indicate the area of cell nuclei. B) Optical section imageapproximately 100 μm from the surface. YFP-paxillin fluorescenceappears to be associated with filament-like structures indicatingcell growth along fibers of the scaffold. (C) Higher-magnificationfluorescence image of REF YFP-paxillin cells on 2d substrate (focaladhesion sites; green) that were stained with DAPI (nuclei; blue)and RFP (stress fibers; red). (D) Optical section image approximately50 μm from the surface, showing a mesh of actin (red) ratherthan stress fibers and smaller clusters of YFP-paxillin compared tothe 2D substrate, which appear yellow due to overlap with red actinfluorescence. E) Bright field image of a 9 μm paraffin thinsection from a position about 0.4 mm below the AG surface. Embeddedin wax cells cannot be distinguished from the paraffin background.(F–I) Haematoxylin and eosin staining makes REF52 YFP Pax cellsvisible by coloring the nuclei blue (hematoxylin) and the cytosolpink (eosin). Due to vigorous dewaxing and staining treatment, theoriginal AG section is highly fragmented. Nevertheless, higher-magnificationreveals cells that are well-interfaced with AG filaments and illustratemorphologies typical for fibroblasts. Scale bars: 10 μm.
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fig4: (A)YFP fluorescence (green) images of REF YFP-paxillin cells on a flatsubstrate, which have grown to a confluent layer. Bright fluorescentspots indicate focal adhesions of the cells. Intracellular homogeneousfluorescence originates in part from cytosolic paxillin. Dark circularregions indicate the area of cell nuclei. B) Optical section imageapproximately 100 μm from the surface. YFP-paxillin fluorescenceappears to be associated with filament-like structures indicatingcell growth along fibers of the scaffold. (C) Higher-magnificationfluorescence image of REF YFP-paxillin cells on 2d substrate (focaladhesion sites; green) that were stained with DAPI (nuclei; blue)and RFP (stress fibers; red). (D) Optical section image approximately50 μm from the surface, showing a mesh of actin (red) ratherthan stress fibers and smaller clusters of YFP-paxillin compared tothe 2D substrate, which appear yellow due to overlap with red actinfluorescence. E) Bright field image of a 9 μm paraffin thinsection from a position about 0.4 mm below the AG surface. Embeddedin wax cells cannot be distinguished from the paraffin background.(F–I) Haematoxylin and eosin staining makes REF52 YFP Pax cellsvisible by coloring the nuclei blue (hematoxylin) and the cytosolpink (eosin). Due to vigorous dewaxing and staining treatment, theoriginal AG section is highly fragmented. Nevertheless, higher-magnificationreveals cells that are well-interfaced with AG filaments and illustratemorphologies typical for fibroblasts. Scale bars: 10 μm.

Mentions: Next, we explored the colonizationdepth within AG scaffolds using inherently fluorescent REF52 cells,which express YFP-paxillin. Paxillin is mainly located in the focalcontacts formed by fibroblast upon adhesion (Figure 4A). Despite the extreme low density and openporous structure, AG scaffolds are opaque and have tremendous lightabsorbing capacities.15 Thus, fluorescenceimaging proved most challenging and required extended illuminationtimes of up to 5 s/frame. Optical image stacks were recorded up toa maximum penetration depth of 300 μm from the surface. Figure 4B shows an opticalsection image taken approximately 100 μm from the surface ofan AG scaffold. In the image AG filaments are well visible as blackfibers, as they absorbed all emitted light; fluorescence signals fromembedded cells indicate progressive growth into the matrix. Figures 4C, D compare actin(red) and paxillin (green) distributions on 2D glass and in 3D AG.On a 2D substrate, actin assembles in stress fibers, and separatefocal adhesion sites are clearly visible. In 3D, paxillin clustersare much smaller and actin forms more of a mesh. This is in agreementwith previous studies that have shown that adhesion structures arequite different in 2D and 3D.6,27−29


ATunable Scaffold of Microtubular Graphite for 3D Cell Growth
(A)YFP fluorescence (green) images of REF YFP-paxillin cells on a flatsubstrate, which have grown to a confluent layer. Bright fluorescentspots indicate focal adhesions of the cells. Intracellular homogeneousfluorescence originates in part from cytosolic paxillin. Dark circularregions indicate the area of cell nuclei. B) Optical section imageapproximately 100 μm from the surface. YFP-paxillin fluorescenceappears to be associated with filament-like structures indicatingcell growth along fibers of the scaffold. (C) Higher-magnificationfluorescence image of REF YFP-paxillin cells on 2d substrate (focaladhesion sites; green) that were stained with DAPI (nuclei; blue)and RFP (stress fibers; red). (D) Optical section image approximately50 μm from the surface, showing a mesh of actin (red) ratherthan stress fibers and smaller clusters of YFP-paxillin compared tothe 2D substrate, which appear yellow due to overlap with red actinfluorescence. E) Bright field image of a 9 μm paraffin thinsection from a position about 0.4 mm below the AG surface. Embeddedin wax cells cannot be distinguished from the paraffin background.(F–I) Haematoxylin and eosin staining makes REF52 YFP Pax cellsvisible by coloring the nuclei blue (hematoxylin) and the cytosolpink (eosin). Due to vigorous dewaxing and staining treatment, theoriginal AG section is highly fragmented. Nevertheless, higher-magnificationreveals cells that are well-interfaced with AG filaments and illustratemorphologies typical for fibroblasts. Scale bars: 10 μm.
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fig4: (A)YFP fluorescence (green) images of REF YFP-paxillin cells on a flatsubstrate, which have grown to a confluent layer. Bright fluorescentspots indicate focal adhesions of the cells. Intracellular homogeneousfluorescence originates in part from cytosolic paxillin. Dark circularregions indicate the area of cell nuclei. B) Optical section imageapproximately 100 μm from the surface. YFP-paxillin fluorescenceappears to be associated with filament-like structures indicatingcell growth along fibers of the scaffold. (C) Higher-magnificationfluorescence image of REF YFP-paxillin cells on 2d substrate (focaladhesion sites; green) that were stained with DAPI (nuclei; blue)and RFP (stress fibers; red). (D) Optical section image approximately50 μm from the surface, showing a mesh of actin (red) ratherthan stress fibers and smaller clusters of YFP-paxillin compared tothe 2D substrate, which appear yellow due to overlap with red actinfluorescence. E) Bright field image of a 9 μm paraffin thinsection from a position about 0.4 mm below the AG surface. Embeddedin wax cells cannot be distinguished from the paraffin background.(F–I) Haematoxylin and eosin staining makes REF52 YFP Pax cellsvisible by coloring the nuclei blue (hematoxylin) and the cytosolpink (eosin). Due to vigorous dewaxing and staining treatment, theoriginal AG section is highly fragmented. Nevertheless, higher-magnificationreveals cells that are well-interfaced with AG filaments and illustratemorphologies typical for fibroblasts. Scale bars: 10 μm.
Mentions: Next, we explored the colonizationdepth within AG scaffolds using inherently fluorescent REF52 cells,which express YFP-paxillin. Paxillin is mainly located in the focalcontacts formed by fibroblast upon adhesion (Figure 4A). Despite the extreme low density and openporous structure, AG scaffolds are opaque and have tremendous lightabsorbing capacities.15 Thus, fluorescenceimaging proved most challenging and required extended illuminationtimes of up to 5 s/frame. Optical image stacks were recorded up toa maximum penetration depth of 300 μm from the surface. Figure 4B shows an opticalsection image taken approximately 100 μm from the surface ofan AG scaffold. In the image AG filaments are well visible as blackfibers, as they absorbed all emitted light; fluorescence signals fromembedded cells indicate progressive growth into the matrix. Figures 4C, D compare actin(red) and paxillin (green) distributions on 2D glass and in 3D AG.On a 2D substrate, actin assembles in stress fibers, and separatefocal adhesion sites are clearly visible. In 3D, paxillin clustersare much smaller and actin forms more of a mesh. This is in agreementwith previous studies that have shown that adhesion structures arequite different in 2D and 3D.6,27−29

View Article: PubMed Central - PubMed

ABSTRACT

Aerographite(AG) is a novel carbon-based material that exists as a self-supportive3D network of interconnected hollow microtubules. It can be synthesizedin a variety of architectures tailored by the growth conditions. Thisflexibility in creating structures presents interesting bioengineeringpossibilities such as the generation of an artificial extracellularmatrix. Here we have explored the feasibility and potential of AGas a scaffold for 3D cell growth employing cyclic RGD (cRGD) peptidescoupled to poly(ethylene glycol) (PEG) conjugated phospholipids forsurface functionalization to promote specific adhesion of fibroblastcells. Successful growth and invasion of the bulk material was followedover a period of 4 days.

No MeSH data available.


Related in: MedlinePlus