Conductive Graphene/Carbon Nanofiber Composite Scaffold, its use for neural tissue engineering and a method of preparation thereof

Abstract

Porous electrically conductive graphene/carbon nanofiber bio-scaffolds can be fabricated having a Youngs modulus and electrical conductivity that both match typical values for brain tissue. Such scaffolds have been tested and found to provide proved differentiation of stem cells into functional neural cells relative to conventional scaffolds. In preferred embodiments, neural cell differentiation in conductive graphene/carbon nanofiber scaffolds is promoted by an applied electrical stimulus.

Claims

1. A method of making an electrically conductive graphene/carbon nanofiber composite scaffold, the method comprising: preparing a suspension of graphene oxide and carbon nanofibers; sonicating the suspension to ensure a homogeneous mixture of the graphene oxide and carbon nanofibers; and reducing the graphene oxide in the suspension with one or more reducing agents to provide an electrically conductive graphene/carbon nanofiber composite scaffold via a sol-gel transition.

2. The method of claim 1, wherein the graphene oxide is prepared by reacting graphite with an oxidizer in an acidic solution.

3. The method of claim 1, further comprising degassing the suspension after the sonicating and prior to the reducing.

4. The method of claim 1, wherein pores of the scaffold have pore diameters in a range from 15 m to 70 m.

5. The method of claim 1, wherein a Young's modulus of the scaffold is between 3 kPa and 7 kPa.

6. The method of claim 1, wherein an electrical conductivity of the scaffold is between 0.1 S/m and 0.3 S/m.

7. A method of directing stem cell differentiation, the method comprising: providing a scaffold made according to the method of claim 1; and exposing the scaffold to a culture medium comprising stem cells for a period of time sufficient to allow the stem cells to differentiate into cells of interest.

8. The method of claim 7, wherein differentiation of the stem cells into cells of interest in the culture medium is driven by an external electrical stimulation.

9. The method of claim 1, wherein an X-ray photoelectron spectrum of the scaffold has an SP C peak that is larger than its CO peak.

10. A scaffold comprising: an electrically conductive graphene/carbon nanofiber composite scaffold; wherein the electrically conductive graphene/carbon nanofiber composite scaffold is porous with a 3-D network of pores.

11. The scaffold of claim 10, wherein pores of the scaffold have pore diameters in a range from 15 m to 70 m.

12. The scaffold of claim 10, wherein a Young's modulus of the scaffold is between 3 kPa and 7 kPa.

13. The scaffold of claim 10, further comprising differentiated stem cells disposed in the scaffold.

14. The scaffold of claim 10, wherein an electrical conductivity of the scaffold is between 0.1 S/m and 0.3 S/m.

15. The scaffold of claim 10, wherein an X-ray photoelectron spectrum of the scaffold has an SP.sup.2 C peak that is larger than its CO peak.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0010] FIGS. 1A-B schematically show use of a bio-scaffold to expedite differentiation of stem cells into neural cells.

[0011] FIGS. 2A-B are images showing two stages of an exemplary fabrication sequence for conductive graphene/carbon nanofiber scaffolds.

[0012] FIG. 3A shows stiffness of conductive graphene/carbon nanofiber scaffolds vs. ratio of carbon nanofibers to graphene oxide.

[0013] FIG. 3B shows electrical conductivity of conductive graphene/carbon nanofiber scaffolds compared to that of other scaffold materials.

[0014] FIGS. 4A-C are SEM (scanning electron microscope) images from a fabricated conductive graphene/carbon nanofiber scaffold.

[0015] FIG. 5 is a histogram of the pore size from a sample of a conductive graphene/carbon nanofiber scaffold.

[0016] FIG. 6 shows FTIR (Fourier Transform InfraRed) spectra from graphite, graphene oxide and a fabricated conductive graphene/carbon nanofiber scaffold.

[0017] FIG. 7A shows XPS (X-ray photoelectron Spectroscopy) data from a graphene oxide sample.

[0018] FIG. 7B shows XPS data from a fabricated conductive graphene/carbon nanofiber scaffold sample.

[0019] FIGS. 8A-B show neural cell differentiation results for various scaffold materials and conditions.

DETAILED DESCRIPTION

Introduction

[0020] To resolve the above-described problem, electrically conductive scaffolds based on graphene oxide and carbon nanofiber (CNF) were prepared by a sol-gel transition associated with a chemical reduction method. Mechanical properties including its modulus have been controlled due to the presence of carbon nanofibers. The addition of carbon nanofibers created a soft conductive scaffold which fully mimics the microenvironment of brain tissue. This work provides a conductive bio-scaffold which overcomes the drawbacks of conventional inert scaffolds in neural tissue engineering.

[0021] FIGS. 1A-B schematically show use of a bio-scaffold to expedite differentiation of stem cells into neural cells. FIG. 1A schematically shows stem cells 106, 108, 110 infiltrated into a pore 104 in a scaffold 102. The desired result of this is shown on FIG. 1B, where the stem cells have differentiated into neural cells 114, 116, 118 having functional structures such as dendrites 122, 124, 126 and axons 132 and 134. Preferably, as shown, axons and dendrites are functionally connected from one neural cell to another. Stimulation 112 can be applied to facilitate this process of differentiation. In general, the success or failure of this kind of approach will depend critically on the properties of the scaffold, and in particular on how well the relevant properties of brain tissue are reproduced by the scaffold. As indicated above, we have found that providing a close match for both mechanical stiffness (i.e., Young's modulus) and electrical conductivity leads to improved neural cell differentiation compared to conventional scaffold materials.

[0022] One embodiment of the invention is a method of making an electrically conductive graphene/carbon nanofiber composite scaffold. The method includes the following steps:

[0023] 1) preparing a suspension of graphene oxide and carbon nanofibers;

[0024] 2) sonicating the suspension to ensure a homogeneous mixture of the graphene oxide and carbon nanofibers; and

[0025] 3) reducing the graphene oxide in the suspension with one or more reducing agents to provide an electrically conductive graphene/carbon nanofiber composite scaffold via a sol-gel transition. A detailed example is described below in the methods section.

[0026] Some general features of preferred embodiments are described here. These features can be included in the above method individually or in any combination. The graphene oxide can be prepared by reacting graphite with an oxidizer in an acidic solution. The suspension can be degassed after the sonicating of step 2 and prior to the reducing of step 3. Pores of the scaffold can have pore diameters in a range from 15 m to 70 m. The Young's modulus of the scaffold is preferably between 3 kPa and 7 kPa. The electrical conductivity of the scaffold is preferably between 0.1 S/m and 0.3 S/m. The X-ray photoelectron spectrum of the scaffold preferably has an SP C peak that is larger than its CO peak. This result is a signature of full reduction of graphene oxide to graphene in the sample.

[0027] Another embodiment of the invention is a method of directing stem cell differentiation. Here the steps include providing a scaffold made as described above, and exposing the scaffold to a culture medium comprising stem cells for a period of time sufficient to allow the stem cells to differentiate into cells of interest. The differentiation of the stem cells into cells of interest in the culture medium can be driven by one or more external stimulations (e.g., 112 on FIG. 1B). The external stimulations are preferably electrical stimulation.

[0028] A further embodiment of the invention is an electrically conductive graphene/carbon nanofiber composite scaffold, where the electrically conductive graphene/carbon nanofiber composite scaffold is porous with a 3-D network of pores. Here composite refers to the carbon nanofibers being physically incorporated into the scaffold without being chemically bonded to the graphene.

[0029] Some general features of preferred embodiments are described here. These features can be included in the above scaffold individually or in any combination. Pores of the scaffold can have pore diameters in a range from 15 m to 70 m. The Young's modulus of the scaffold is preferably between 3 kPa and 7 kPa. The electrical conductivity of the scaffold is preferably between 0.1 S/m and 0.3 S/m. The X-ray photoelectron spectrum of the scaffold preferably has an SP.sup.2 C peak that is larger than its CO peak. This result is a signature of full reduction of graphene oxide to graphene in the sample. Differentiated stem cells can be disposed in the scaffold.

Experimental Example

[0030] FIGS. 2A-B are photographs of conductive graphene scaffold (CGS) before chemical reduction (Sol-status, (FIG. 2A)) and after chemical reduction (Gel-status, (FIG. 2B)).

[0031] FIG. 3A shows how addition of different ratio of carbon nanofibers can modulate its stiffness. FIG. 3A is a bar graph showing Young's modulus (ranging from 12 kPa (No addition of carbon nanofibers, Stiff-CGS) to 3 kPa (GO:CNFs, 1:1, Soft-CGS)) with different ratio of carbon nanofibers in the scaffolds. Data are presented as meanstandard deviation.

[0032] FIG. 3B shows electrical conductivity of scaffolds of this work as compared to the electrical conductivity of other scaffold materials. The conductivity and elastic modulus can mimic physiological brain, which we have found to provide rapid neuronal conversion of stem cells into functional neurons.

[0033] FIG. 4A is an image of surface morphology of a fabricated conductive graphene scaffold showing its porous structure. The scale bar indicates 200 m.

[0034] FIG. 4B is a cross sectional image showing the porous structure inside the conductive graphene scaffold. The scale bar indicates 200 m.

[0035] FIG. 4C is an image of carbon nanofibers intertwined with reduced graphene oxides (rGOs) inside the conductive graphene scaffold. The scale bar indicates 400 m.

[0036] FIG. 5 is a histogram of pore size distribution in a fabricated conductive graphene scaffold. The average pore size in this sample was 31.3 m and the standard deviation was 2.3 m. Here pore size is the pore diameter, as opposed to the pore radius.

[0037] FIG. 6 shows FT-IR (Fourier Transform InfraRed spectroscopy) analysis of different materials. GO (graphene oxide) was fully reduced by reducing agents, forming the conductive graphene scaffold. In presence of carbon nanofibers, the soft conductive graphene scaffold sample (Soft-CGS) showed negligible oxide groups, such as OH (3200 cm-1) and COOH (1700 cm-1). These results indicate that the fabrication of the conductive graphene scaffold was achieved by chemical reduction which induced a sol-gel transition of the solution.

[0038] FIGS. 7A-B show results of XPS (X-ray Photoelectron Spectroscopy) analysis probing the mechanism of gelation during conductive graphene scaffold synthesis, where graphene oxide was reduced in presence of reducing agents. FIG. 7A shows the graphene oxide XPS results, which include peaks from oxide groups such as CO and OCO as labeled on the plot. FIG. 7B shows the conductive graphene scaffold XPS results. Here the conductive graphene scaffold didn't have high CO and OCO bands. Exceptionally, the conductive graphene scaffold showed high peak of SP2 carbon band, indicating that while reducing oxides, reduced graphene oxides were intertwined with carbon nanofibers due to hydrophobic interaction, eventually forming a gelled conductive graphene scaffold.

[0039] FIGS. 8A-B are bar plots showing the proportion of TUJ1.sup.+ (FIG. 8A) and MAP2.sup.+ (FIG. 8B) cells (neuronal markers) differentiated from human-induced pluripotent stem cells at 14 days with conditions as described below in the methods section. Here TUJ1 is short for Neuron-specific Class III beta-tubulin and MAP2 is short for Microtubule associated protein-2. The soft conductive graphene scaffold that was electrically stimulated showed more rapid maturation of stem cells into neurons than other conditions as well as more rapid maturation than has been reported in the literature using chemical factors. Note that TUJ1 is an earlier marker of neurons, but MAP2 is post-mitotic stage marker of neurons. Thus, cells could have TUJ1+ cells with smaller population of MAP2+ cells. In other words, the markers are not exclusive so cells can show both markers, but the ones with MAP2 are more mature.

Methods:

[0040] 1) Preparation of graphene oxides (GOs): 1 g of graphite flakes (Ashbury Carbon, N.J.) supplied from Ashbury Carbons (Asbury, N.J.) was added into acidic solution (9:1, H.sub.2SO.sub.4:HNO.sub.3) and the solution was stirred without heat for 30 min. 6 g of potassium permanganate was slowly added into the solution, and the solution was covered with tin foil, heat to 50 C., and incubate overnight. 5 mL of H.sub.2O.sub.2 ice-cold solution was added and stirred for 2 hours until no bubbles found. The solution was centrifuged at 4,500 rpm for 45 min. The purification with HCl (0.1 M) and three consecutive washing with DI H.sub.2O was applied to remove unreacted carbon and metal ions. Collected graphene oxides (10 mg/ml) was kept at 4 C. until further usage.

[0041] 2) Preparation of 3D-nanoconfined conductive graphene scaffold (3D CGS): Carbon nanofibers were purchased from Sigma Aldrich, and had diameter 100 nm and length 20-200 m (manufacturer's specifications). Equal amount of carbon nanofibers were suspended in graphene oxide solution to a concentration of 0.2 wt %. Suspensions were sonicated using a bath sonicator (Branson Ultrasonic) for 1 hour at 60 W followed by centrifugation at 2,000 rpm for 20 min (Beckman Coulter Allegra 25R) to sediment carbon nanofiber bundles. We degassed the concentrated GO:CNF suspensions to remove any bubbles since bubbles significantly degrade the mechanical integrity of 3D CGS. Reducing agents including sodium iodide and ascorbic acid to a concentration of 10 wt % to induce self-assembly of 3D CGS were added into suspensions and it was poured into cylindrical molds. The suspensions formed 3D CGS via solution to gelation transition (sol-gel transition) at 80 C. within 24 hours due to van der Walls interactions between reduced-GOs/CNFs in presence of reducing agents. 3D CGS had dimensions of 62 mm (diameterheight). The 3D CGSs were neutralized by washing with deionized water until pH equilibrated to 7. Collected 3D CGSs were autoclaved and was kept at 4 C. until further usage.

[0042] 3) Differentiation of neural progenitor cells (NPCs) to neurons using electrical stimulation: NPCs were plated on CGS (100,000 cells/cm.sup.2) previously coated with 10 g/ml poly-L-ornithine solution (Sigma-Aldrich, St. Louis, Mo.) and 4 g/ml laminin (Sigma-Aldrich, St. Louis, Mo.) in PBS (phosphate-buffered saline). Then scaffolds with the cells were incubated under standard cell culture conditions (37 C., 5% CO.sub.2) for 24 hr. In vitro iPSC-electrical stimulation was applied by means of an alternating current-electrical stimulation. Here iPSC is short for induced pluripotent stem cells. The chamber includes parallel ITO (indium tin oxide) patterned-electrodes, separated by a distance of 3 mm. The electrodes were connected to a waveform generator (Keysight, Englewood, Colo.). The cells cultured on CGS were exposed to electrical stimulation for 1 hr. After the stimulation was applied, the scaffolds were carefully transferred to a 24-well plate with 1 mL of fresh medium. On day 7, cells were fixed with 4% paraformaldehyde (Electron Microscopy Sciences) for 1 min and then permeabilized and blocked with blocking buffer (0.1% Triton X-100, 1% BSA (Fisher BioReagents, Santa Clara, Calif.), 5% normal goat serum (NGS, Invitrogen, Waltham, Mass.) for 1 hr at room temperature. Primary antibodies (TUJ1 And MAP2) were incubated in blocking buffer at 4 C. overnight, followed by three 15-min PBS washes and detected by secondary antibodies (Alexa Flour 488, 555 or 647, Life Technologies). Samples were counter-stained with DAPI (Sigma-Aldrich, St. Louis, Mo.) to visualize nuclei and mounted with Fluoromount Aqueous Medium (Sigma-Aldrich, St. Louis, Mo.) before imaging. Samples were imaged on a Keyence All-in-One Fluorescence Microscope (BZ-X700) (Keyence corp., Itasca, Ill.) using 20 objectives. Image of cells on CGSs are presented as maximum intensity projections of z-stacks generated from BZ-X Analyzer. The neuronal differentiation efficacy of iPSCs was quantified by counting the total number of TUJ1-positive cells with neuronal morphology. The number of TUJ1-positive cells was divided by the total number of cell nuclei (DAPI-positive) to demonstrate the percentage of neuronal differentiation. Calculations were performed using randomly selected 4 different locations from 4 individual samples at 20 magnification.