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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.


SEM imagesof REF 52 cells after 4 days of growth within cRGD functionalizedAG with a 4:1 mixture of DSPE-PEG2000-NH2/ DSPE-PEG2000-cRGD.(A) The medium sized overview scan shows growth of numerous cells(arrows) along fibers in different planes within the 3D network. (B–D)Zoom-in on the interface between cell and functionalized AG surfaceshow a tight physical connection between cells and scaffold material.(E) Results of MTT-Formazan absorbance measurement, showing mean valuesof cell viability (two independent experiments, three technical repeatsin each of them) and ± standard deviation for REF 52 cells treatedwith extracts of pristine (AG) and PEG-lipid conjugated (AG PEG-lipid)aerographite, as well as normal medium (untreated) and 15% DMSO (positive).
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fig3: SEM imagesof REF 52 cells after 4 days of growth within cRGD functionalizedAG with a 4:1 mixture of DSPE-PEG2000-NH2/ DSPE-PEG2000-cRGD.(A) The medium sized overview scan shows growth of numerous cells(arrows) along fibers in different planes within the 3D network. (B–D)Zoom-in on the interface between cell and functionalized AG surfaceshow a tight physical connection between cells and scaffold material.(E) Results of MTT-Formazan absorbance measurement, showing mean valuesof cell viability (two independent experiments, three technical repeatsin each of them) and ± standard deviation for REF 52 cells treatedwith extracts of pristine (AG) and PEG-lipid conjugated (AG PEG-lipid)aerographite, as well as normal medium (untreated) and 15% DMSO (positive).

Mentions: REF52 were cultured for 4 days, then fixed with paraformaldehydeand prepared for SEM imaging by CPD. A thin layer of gold was appliedfor visualization of cells within the scaffolds and reduction of thedestructive influence of the electron beam on biological samples.SEM images of fibroblasts near the surface of the AG bulk material(Figure 3) revealedthe typical polygonal cell shape with elongated cytoplasm projectionsattaching to the scaffold. Images at higher magnification (Figure 3C,D) showed contactformation of the plasma membrane with the AG surface indicating theability of cRGD functionalized AG to promote integrin mediated specificcell adhesion. The viability of cells upon exposure to pristine andDSPE-PEG2000-NH2 functionalized AG was tested accordingto the norm ISO 10993, which proposes standardized conditions forbiological evaluation of medical devices and materials. In particular,assay protocols outlined in parts 5 (ISO 10993–5:2009) and12 (ISO 10993–12:2004) of this norm were applied. Briefly,REF52 cells were cultured for 24 h in extract medium that had beenincubated with AG and PEG-lipid conjugated AG at 37 °C for 72h. To determine cell viability the colorimetric MTT metabolic activityassay was used with cells incubated in untreated medium as negativecontrol and cells incubated in 15% DMSO as positive control (Figure 3E). The results werenormalized to the viability of the negative control and show neithera negative effect of functionalized AG nor pristine AG on REF 52 cells.


ATunable Scaffold of Microtubular Graphite for 3D Cell Growth
SEM imagesof REF 52 cells after 4 days of growth within cRGD functionalizedAG with a 4:1 mixture of DSPE-PEG2000-NH2/ DSPE-PEG2000-cRGD.(A) The medium sized overview scan shows growth of numerous cells(arrows) along fibers in different planes within the 3D network. (B–D)Zoom-in on the interface between cell and functionalized AG surfaceshow a tight physical connection between cells and scaffold material.(E) Results of MTT-Formazan absorbance measurement, showing mean valuesof cell viability (two independent experiments, three technical repeatsin each of them) and ± standard deviation for REF 52 cells treatedwith extracts of pristine (AG) and PEG-lipid conjugated (AG PEG-lipid)aerographite, as well as normal medium (untreated) and 15% DMSO (positive).
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fig3: SEM imagesof REF 52 cells after 4 days of growth within cRGD functionalizedAG with a 4:1 mixture of DSPE-PEG2000-NH2/ DSPE-PEG2000-cRGD.(A) The medium sized overview scan shows growth of numerous cells(arrows) along fibers in different planes within the 3D network. (B–D)Zoom-in on the interface between cell and functionalized AG surfaceshow a tight physical connection between cells and scaffold material.(E) Results of MTT-Formazan absorbance measurement, showing mean valuesof cell viability (two independent experiments, three technical repeatsin each of them) and ± standard deviation for REF 52 cells treatedwith extracts of pristine (AG) and PEG-lipid conjugated (AG PEG-lipid)aerographite, as well as normal medium (untreated) and 15% DMSO (positive).
Mentions: REF52 were cultured for 4 days, then fixed with paraformaldehydeand prepared for SEM imaging by CPD. A thin layer of gold was appliedfor visualization of cells within the scaffolds and reduction of thedestructive influence of the electron beam on biological samples.SEM images of fibroblasts near the surface of the AG bulk material(Figure 3) revealedthe typical polygonal cell shape with elongated cytoplasm projectionsattaching to the scaffold. Images at higher magnification (Figure 3C,D) showed contactformation of the plasma membrane with the AG surface indicating theability of cRGD functionalized AG to promote integrin mediated specificcell adhesion. The viability of cells upon exposure to pristine andDSPE-PEG2000-NH2 functionalized AG was tested accordingto the norm ISO 10993, which proposes standardized conditions forbiological evaluation of medical devices and materials. In particular,assay protocols outlined in parts 5 (ISO 10993–5:2009) and12 (ISO 10993–12:2004) of this norm were applied. Briefly,REF52 cells were cultured for 24 h in extract medium that had beenincubated with AG and PEG-lipid conjugated AG at 37 °C for 72h. To determine cell viability the colorimetric MTT metabolic activityassay was used with cells incubated in untreated medium as negativecontrol and cells incubated in 15% DMSO as positive control (Figure 3E). The results werenormalized to the viability of the negative control and show neithera negative effect of functionalized AG nor pristine AG on REF 52 cells.

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.