Investigation of the Effect of Local Electrical Stimulation on Cells Cultured on Conductive Single-Walled Carbon Nanotube/Albumin Films ()
1. Introduction
For the past five decades electrical stimulation effect on accelerated regeneration of bone tissues, skin injury repair and targeted drug delivering has been widely investigated [1]. The main problem with conventional methods of supplying an external electric field is that it is impossible to localize the field in the healing area. In general case a considerable area of the body is exposed to electrical stimulation. Extending the work area and the distance between electrodes and the healing area implies increasing of electrical field intensity, required for stimulating individual cells. This problem may be solved by using implanted electrodes localizing the electric field in the necessary area of the body. In this case, standard materials which are used for electrodes, for example, stainless steel, platinum or titanium can be replaced by nonmetallic, more biocompatible and conductive materials [2].
Carbon nanotubes, in particular, are one of promising nanotechnology products, and since their structure is geometrically close to collagen [3]—the main protein of the mammal connective tissue, they can be used in biological engineering as a scaffold material for tissue regeneration [4]. Unique electronic properties, high mechanical strength, excellent flexibility and large specific surface area of nanotubes make them suitable for creating novel biocompatible composite materials for tissue engineering [5].
Producing a scaffold material is widely investigated in cell seeding and growing applications [6]. At present, there are a number of works on producing composite materials based on nanotubes for bioengineering, and namely, for bone and cartilaginous tissue regeneration [7,8], and fibroblast growth investigation [9].
In osteoblast growth an additional possibility of delivering an electrical signal to cells through the culture medium and current-conductive composite containing nanotubes is used. In this case, a 46% increase in proliferation [10] was observed after electrical stimulation. Various applications of nanotubes and current-conductive composites based on them in neurosurgery [11] are also considered. For further developments in integrating nanotubes in living tissues, a number of serious problems like improving the biocompatibility and biodegradation of nanotubes as well as producing composites based on protein matrices must be solved [12]. Nevertheless the correlation analysis of the local pulse electric field stimulation effect on proliferation and electric fields mediate motility (galvanotaxis) on nanotube substrates has not been done so far.
This work suggests the development of composite based on single-walled carbon nanotubes (SWNT) and bovine serum albumin (BSA) to form conductive substrates. A device for local field supply to cells through nanosized electrodes has been developed. Spatial organization and proliferation of human embryonic fibroblasts (HEF) cultured in vitro under the influence of a pulsed local electric field are studied. The research shows that by stimulating cells through nanosized electrodes at low amplitudes of electric field intensity and small signal oscillation rates proliferation raises by 26%. Whereas increasing the field strength leads to motility and HEF cell reorganization according to electric field gradient.
2. Materials and Methods
2.1. Choosing the Material
Most commonly the material of carbon nanotubes obtained by arch discharge or laser evaporation of graphite or by chemical decomposition of carbonaceous vapor is a powder that should be purified, filtered from undesirable impurities (such as amorphous carbon, defective nanotubes, catalyst particles), as well as functionalized, in some cases, to obtain nanotube properties required for the specific application. However in this case, preparation of solutions is needed. It is well-known that nanotubes can be dissolved in most polar and nonpolar solvents, so to dissolve SWNTs obtained in the arc discharge, it is necessary to split bunches and bundles of them which were formed in the process of growth. The most effective way to do that is additional functionalizetion of nanotubes by surfactant materials (SM), which allows a stable colloidal solution of single nanotubes of any concentration to be achieved. These SM solutions are ideal to form thin percolated films on any surfaces. By regulating nanotube concentration in the solution one can regulate the thickness of the film to be formed on the surface. As we have shown previously [13], the nanotube orientation and density on the surface can be controlled while using different deposition techniques: dipping, pulling, centrifugation, sputtering.
To prepare conductive composites based on nanotubes one can use their mixtures with proteins as a natural polymer providing a biocompatible substrate and improved adhesion of cells [12]. Albumin is one of the most important transport proteins, which regulates metabolism functions in cell growth and tissue regeneration. In this research we suggest using BSA protein molecules as a surfactant to form a biocompatible conductive layer on the cover-slip. Previously, a composite consisting of nanotubes and albumin was examined as a scaffold-forming material in implants in cartilaginous tissue regeneration [14,15].
2.2. Substrate Fabrication
2.5 mg of 99.5 mass % SWNT provided by A. V. Krestinin (Institute of Problems of Chemical Physics of RAS, Moscow, Russia) were placed into a BSA aqueous solution (10 mg of BSA in 5 mL of water) and dispersed in a Branson B300 (Branson Ultrasonics, Danbury, CT, USA) ultrasonic bath (34 kHz, 50 W) for 10 hours. Typical length of nanotubes was less than 1000 nm after ultrasonic treatment. Nevertheless the CNT combines in bundles up to 5 mm long and about 10 nm in diameter. Cover-slips, 24 × 24 mm in size, 0.13 - 0.17 mm thick were preliminary mechanically washed with cotton in 2-propanol, then they were kept in 2-propanol for 15 minutes and placed in ultrasonic bath. On one of the surfaces of the cover-slip two gold contact pads, 30 nm thick, were formed by magnetron sputter deposition (Emitech K575X, Quorum Technologies, Ringmer, UK). About 25 mL of nanotube solution in albumin were applied onto this surface with a microdispenser and a thin film was made to cover the whole slip surface by rodcoating method, then the film dried for 15 minutes at 40˚C. To improve the film adhesion and conductance the structure was annealed in the air at a temperature of 150˚C for 2 minutes. Figure 1 shows the photography of a cover-slip with the current-conductive SWNT/BSA coating formed. The resistance of the structures investigated in the work ranged from 100 kOhm to 10 MOhm, but in the experiment for proliferation estimation at low stimulation voltage we’ve used a set of samples with close resistance (several MOhm).
It should be noted that the samples of SWNT/BSA film obtained not only possessed conductance but also transparency within the visible range at a level of 85% - 90%.
2.3. Electrical Stimulation System
To carry out experiments with electrical stimulation of
Figure 1. Cover-slip with gold electrode system covered by SWNT/BSA film.
cells, a custom system for supplying electrical signals which comprises a 6-well plate, a signal generator and an oscilloscope has been developed. The plate cover has a breadboard with a connector fixed on it. Over each of these 6 wells two sharp needle electrodes with diameter 0.3 mm made of a 40KHNM-VI biocompatible alloy with a gold electrolytic coating (Doriva, Moscow, Russia) were placed. The electrodes were connected in pairs in 2 rows of 3 wells each to provide lead output to the signal generator connector. On the one hand, this ensured biological compatibility with the medium, on the other, the contact area of the whole surface of the needle with the cultural medium was much smaller than the area of the modified cover-slip (~40 times), which minimized the current contribution through the medium. In each set we’ve used 5 wells for experiment and one for control (Figure 2). To apply voltage we used a RIGOL DG1022 generator (Rigol Technologies Inc., USA).
The generator can be set to arbitrarily shaped stimulatory signal, as well as to produce two independent signals in parallel. Due to the modified cultural plate design, the system was able to send a signal directly to the cells through the SWNT/BSA film. The voltage generator was connected with the plate by means of a flexible cable with the core diameter of 0.1 mm, which could be easily placed in the CO2 incubator. Figure 2(b) shows the schematic diagram of the connection.
For each experiment we used five cover-slips covered by SWNT/BSA film: two slips—to minimize statistical error for proliferation measurements, one—for morphology investigation, one—for microscopy analysis, one was cultured without voltage applied and used as control. Also there was one pristine cover slip with voltage applied through the medium.
2.4. Cell Culture
HEF—human embryonic fibroblast cells were provided by the Tissue Cultures Laboratory of Ivanovskiy Institute of Virology, a Federal State Institution of the Ministry of Health and Social Development (Moscow, Russia), were cultured in the electrical stimulation unit (Figure 2(a)). Linbro 6-well plates (MP Biomedicals, Solon, OH, USA) were used as a culture dish. The unit, plates, and coverslips were sterilized in 70% ethanol solution for 10 minutes with the follow-on 20-minute ultraviolet treatment. Immediately prior to the incubation cover-slip samples were rinsed in the Eagle culture medium for 10 min to eliminate contaminants. After removing this medium the HEF cells in the concentration of 105 cells/mL were added into each well in the amount of 1 mL of the Eagle medium with 10% FBS (fetal bovine serum) and incubated for 24 hours in a thermostat with 5% CO2 at 37˚C.
After a 24 hour incubation with no voltage applied, a
Figure 2. Electrical stimulation system schema. (a) A plate of 6-well plate with a system of gold electrodes. The electrodes are placed into the medium to provide a contact with the substrate during electrical stimulation; (b) Schematic diagram of connecting components of the electrical stimulation system.
signal of 5 pulses was sent with 5 ms pulse duration, 5 ms spacing between pulses, 1 s interval between pulse groups. The main pulse characteristics were chosen with previous experiments in electrical stimulation of growing fibroblasts with standard two carbon electrode unit taken into consideration [16]. We have done 3 sets of experiments with different amplitudes 10, 50, 100, 200, 500, 5000 mV applied to the cells. Since the resistance of wires and electrodes is relatively small, the main voltage drop through SWNT/BSA film was expected. Despite the fact that the resistance of the fabricated SWNT/BSA film samples varied, in the experiment, for proliferation estimation we used samples of the same order of resistance (from 3 to 10 MOhms). The electrical stimulation lasted for 48 hours. In addition, we conducted an experiment where the current was applied to needle electrodes placed into culture medium with cover-slips in the absence of a conductive substrate. As expected, the size of the electrodes was relatively small for a significant current to leak through the medium.
2.5. Proliferation and Morphology Investigation
The proliferation was defined by means of a modified MTT assay. For this purpose, the Eagle medium with 10% FBS was removed after electrical stimulation, the cover-slips were moved to a clean plate, where 1 mL of the Eagle medium and 200 ml of MTT solution (with the initial concentration of 5 mg/mL) were added. The coverslips were incubated in the thermostat with CO2 at 37˚C for 4 hours. Then the medium with MTT removed and 1 mL of DMSO was added into each well to dissolve MTT-formazan reduced by the cells. Cell precipitates with MTT-formazan on glass samples were resuspended for 5 min and the solution absorbance was measured by a Titertek Multiscan Plus photometer (Flow Laboratories, Helsinki, Finland) on a 492 nm wavelength. To achieve that, 1 mL of DMSO with the dissolved formazan was transferred to a 96-well plate, with a 100 mL per well dosage. A structure with gold electrodes and a SWNT/ BSA film applied, which was in one of the wells of a 6- well plate, but without voltage supply was used as a control.
The cover-slips with a SWNT/BSA coating were investigated with atomic force microscope Solver P47 (NT-MDT, Moscow, Russia) in a semi-contact mode before and after the cell growth. For AFM measurements samples were removed from culture medium, fixed in 2.5% glutaraldehyde for 30 minutes and washed in the phosphate buffer saline (PBS) 2 - 3 times, 2 minutes each, then dehydrated in 50%, 70% and 96% solution of ethanol for 2 min in each one. Some of the samples after electrical stimulation were stained with azur-eosine for morphology study.
3. Results
First of all we have investigated the quality of film preparation methods. Looking at the surface topography shown at Figure 3 one can say that this method produces a sufficiently uniform film. As nanotubes are surrounded with albumin molecules during water solution preparation, CNT functionalization and additional binding may occur in the process of supersonic treatment [17]. Thus, originating non-covalent bonds between nanotubes and albumin, and CNT functionalization may result in higher
Figure 3. AFM image of a SWNT/BSA film on the coverslip surface after solution deposition and annealing at 100˚C (bar 5 μm). The film thickness is ~100 nm.
uniformity of nanotube distribution in the nanomaterial array, as well as in structuring albumin itself on the nanotube surface [18]. However, it can be seen from the image, that bunches do not split completely: the film consists of bundles up to 10 nm in diameter.
Annealing at 100˚C for 2 minutes increases the conductance of the structures, but we also assume that annealing may not cause the protein denaturation, as SWNTs increase thermal stability of proteins [19].
Studying the morphology of HEF cells grown on glasses with SWNTs after electrical stimulation showed that the culture consisted of fibroblast-like cells with oval nuclei having big nucleoli, 1 - 4 in a nucleus, the cytoplasm was low-reticular, but there were some nanotube clusters varying in form and size (Figure 4). At 100 mV and higher voltages some changes in the cell morphology can be seen. On the whole, one can observe a cluster of cells on spots with thicker nanotube film and their characteristic orientation along the gradient lines of the electric field, while the voltage increases up to 100 mV (Figure 5). More over at voltages higher then 100 mV cell tend to aggregate in large clusters in the middle of the substrate.
AFM investigation was carried out on fixed cells. As nanotubes and cell membranes have different stiffness factors, it was possible to distinguish nanotubes from cells with AFM phase mode. It was found that nanotubes covered the cells on top, especially in the area of cell outgrowth formation (Figure 6). Based on AFM measurements we can suggest that while growing cells not only developed on the surface of the SWNT/BSA composite, but also nanotubes are attaching to the edges of cell’s membrane (Figures 6(a) and (b)), providing better adhesion.
The Figure 6 demonstrate the results for 10 mV pulse electrical stimulation, the same results of nanotube adhesion observed for all range of low voltage applied. On the
Figure 4. Micrographs of HEF cells fixed on the third day after seeding on cover-slips modified with a SWNT/BSA film (magnification ×400). For the last 48 hours culturing took place at 10, 50, 100, 200 mV voltages. On the third day cell processes started to connect, but contact inhibition was absent.
Figure 5. Micrographs of HEF cells fixed on the third day after seeding on cover-slips modified with a SWNT/BSA film (bar 200 μm). The last 48 hours the pulse electric stimulation was applied with amplitude 10 mV (a) and 200 mV (b).
Figure 6. 3D visualization of AFM data (a); close-up view (b); 2D AFM (c) and phase contrast image (d) of HEF cells interaction with nanotubes on a cover-slip modified with a SWNT/BSA film (bar 10 μm).
whole, the AFM topography shows quite good adhesion of cells to the film, which indirectly confirms biological compatibility of the material.
Figure 7 shows the MTT-test absorbance for cells grown at different stimulation signal applied. It can be