A Tunable Scaffold of Microtubular Graphite for 3D Cell Growth

Aerographite (AG) is a novel carbon-based material that exists as a self-supportive 3D network of interconnected hollow microtubules. It can be synthesized in a variety of architectures tailored by the growth conditions. This flexibility in creating structures presents interesting bioengineering possibilities such as the generation of an artificial extracellular matrix. Here we have explored the feasibility and potential of AG as a scaffold for 3D cell growth employing cyclic RGD (cRGD) peptides coupled to poly(ethylene glycol) (PEG) conjugated phospholipids for surface functionalization to promote specific adhesion of fibroblast cells. Successful growth and invasion of the bulk material was followed over a period of 4 days.

S-3 centrifugal filters at 10,000 g for 120 min to remove carbonate buffer followed by 3 washing steps with ultrapure water (200 µl) at 10,000 g for 40 min for purification of the resulting DSPE-PEG2000-cRGD. The recovered concentrated cRGD-PEG-lipids were stored at -20°C until use.
Cell culture and cell seeding: Rat embryonic fibroblasts (REF52) and fluorescent rat embryonic fibroblasts (REF52 YFP Pax), were cultured in Dulbecco's modified Eagle's medium (DMEM; Gibco Laboratories) supplemented with 10% fetal calf serum, 1% penicillin/streptomycin, and 2 mM L-glutamine (all from Gibco Laboratories) in an incubator at 37°C and 5% CO 2 level. About 20,000 cells were seeded on functionalized AG and 10,000 cells were seeded into empty control wells. Growth was visually inspected until control cells reached confluence after day 4. Then the cells were fixed with 4% paraformaldehyde (PFA) in Hank's buffered salt solution (both Sigma) for 20 min and washed with PBS.
Actin fiber and nuclear staining with Phalloidin and Hoechst: PFA fixed control cells and AG/cell samples were washed with PBS three times and 0.1% Triton X-100 in PBS was added for 5 min followed by three times washing with PBS. Next 20 µl PBS and 5 µl Phalloidin and 0.2 µl Hoechst (10 µg/ml) were mixed. Phalloidin stains actin fibers, and Hoechst stains the nucleus of a cell. Four µl of staining solution were added to each sample and incubated for 20 min at 37 °C. Afterward, the solution was removed and the wells were washed with PBS three times and transferred to an IX81 microscope to examine them.
Fluorescence microscopy: Fluorescence imaging was done on an Olympus IX81 inverted microscope equipped with a metal halide lamp, and a monochrome CCD camera (Hamamatsu C9300). In order to observe the different fluorescently labeled cell features the following filters were used: YFP to observe YFP conjugated paxillin that marks adhesion complexes, DAPI for nuclear staining with Hoechst, and RFP to visualize phalloidin stained actin fibers. Setup control S-4 and image recording were assisted by Olympus CellSense Dimension software. Control cells were recorded in epi-fluorescence configuration at 0.1 sec illumination. Images of AG bulk samples were taken as optical image stacks of 20 to 80 sections in epi-fluorescence by motorstage assisted change of the focal plane in 5 µm-steps. Illumination was set to 5 sec per image to provide enough fluorescence signals at reasonable signal to noise ratio. Image processing of monochrome images was done using CellSense Dimension software. Composite images of all three channels were done in Image J with adjustment of contrast and brightness.
MTT based viability assay: In accordance with protocols in part 12 of ISO 10993 (ISO 10993-12:2004) 'Sample preparation and reference materials', we have prepared extracts from three PEG-lipid functionalized and three pristine AG samples in cell culture medium with a mass/volume ratio of sample and medium of 0.1 g/ml taking into consideration the 'standard surface areas and extract liquid volumes' outlined in Table 1  The MTT converted to formazan was extracted by adding isopropanol alcohol, and the optical density of formazan was measured in a plate reader (Bio-Tek instruments, µQuant) (at 570 nm).

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Super critical point drying and scanning electron microscopy: PFA fixed AG/cell samples were dehydrated stepwise in graded ethanol (EtOH) of increasing alcohol concentrations starting at 60% EtOH and incubation period of 30 min followed by 80% (30 min), 90% (30 min), 95% (1 h) and 100% (>1 h). Thereafter, EtOH was substituted against acetone and the samples were transferred into the chamber of the critical point dryer (CPD, BAL-Tec CPD030). For SEM imaging a 7,5 nm layer of gold was applied directly after CPD. Imaging was done in Supra 55VP field emission SEM (Zeiss) applying an acceleration voltage of 2 kV. SEM images have been cropped but are otherwise displayed as recorded. Scale bars have been included using CorelDraw X7.
Preparation of AG sections: PFA fixed AG/cell samples were dehydrated in graded EtOH.
Concentrated EtOH was then substituted against xylene, which was changed 3 times with 1 h settling time for each step. Afterwards samples were soaked in paraffin of 56-58 °C for 1.5 hours, drained and immersed again for embedding into blocks of solidified paraffin. After complete hardening, paraffin was trimmed down to the sample surface and sections of 9 µm thickness were prepared with a microtome. Sections were placed onto glass slides and dried at 37 ºC. Prior to hematoxylin/eosin (HE) staining with Mayer's Haemalaun and 0.1 % Eosin-B solution (both from Merck) paraffin was removed and sections were rehydrated by reversal of the dehydration procedure.
Dewaxing of AG/cell sections and haematoxylin/eosin staining. Dewaxing was done by placing glass slides with paraffin sections in xylene three times for 3 min. Then the glass slides were passed through ethanol solutions of decreasing concentrations (100% EtOH 3 x 3 min, 90% 2 x 3 min, 80% 2 x 3 min, 70% 1 x 3 min, 50% 1 x 3) and finally into deionized water for two S-6 times 3 min. After rehydration glass slides were incubated in Mayer's Haemalaun solution (Merck) for 3 min, rinsed with 0.1% HCl for 2 sec and washed under running water for 1 min.
Next, glass slides were placed in 0.1% Eosin-B solution (Merck) for 3 min followed by 30 sec rinsing with water. Lastly, samples were dehydrated again in an ascending graded ethanol series, soaked in xylene two times, and then covered with Neo-Mount (Merck) and a cover slip.
Optical bright field microscopy was done on an Olympus BX43 equipped with a color CCD camera (ImagingSource). Final image contrast and brightness of presented micrographs were adjusted using CorelDraw X7.

SI APPENDIX I: Surfactants for immersion of AG in aqueous media -test series
A vast number of molecules have been investigated for dispersion of carbon based materials. 1 We have chosen amphiphilic agents that were successfully tested for large diameter CNTs, as AG filaments range from several 100 nm up to a few micrometers. We have tested the following agents at different concentrations in aqueous solution: 1. Bovine serum albumin (BSA), a serum albumin protein derived from cows, which is a standard low cost protein in lab experiments and cell culture, and shows no adverse effects.    The sections were investigated under a light microscope to assess filament thickness, pore size, matrix connectivity, and homogeneity of these parameters ( Figure S5A-C). Sections of the ultralightweight AG C111 generally revealed that the filament lengths and diameters, as well as the pore sizes ranged below typical structural parameters presented by natural extracellular matrix.
Sections of AG C107 showed reduced connectivity of filaments, which might be an artificial result of the harsh treatment during paraffin embedding where paraffin was repeatedly filled and drained into and from the bulk sample, or may be related to structural problems of the ZnO template. Regions with disrupted network connectivity were much fewer in C116 with the S-12 highest specific weight of > 1mg/ccm. Thus we assumed that interfilament connections were more robust, suggesting that this type of AG might be more suitable for future experiments.