CONDUCTIVE SCAFFOLDS FOR GUIDED NEURAL NETWORK FORMATION

Abstract

The present invention relates generally to the manufacture of conductive scaffolds of micro and/or nanofibers with the help of different printing techniques (e.g., near-field electrostatic printing, inkjet printing), such scaffolds enabling the formation of two-dimensional (2D) or three-dimensional (3D) neural networks to mimic the native counterparts. Applications of such patterned conductive scaffolds include, but are not limited to, an engineered conduit for guiding the differentiation and outgrowth of neural cells in peripheral nerve damage or in large-volume spinal cord injury under the electrical stimulation. Meanwhile, the scaffolds could also locally deliver various biomolecules in conjunction with electrical stimulation for facilitated nervous system regeneration (FIG. 1).

Claims

1-21. (canceled)

22. A method for making an electrically-conductive scaffold, comprising the steps of: obtaining a biomimetic matrix; providing said biomimetic matrix with an electrically-conductive pattern of reduced graphene oxide by directly coating said biomimetic matrix with a graphene oxide solution and reducing said graphene oxide solution into said pattern of reduced graphene oxide; and applying a cationic polyelectrolyte to said biomimetic matrix.

23. The method of claim 22, wherein said providing step comprises the steps of loading said graphene oxide solution into a cartridge and printing said graphene oxide solution onto said biomimetic matrix via inkjet printing.

24. (canceled)

25. The method of claim 23, wherein said reducing step is performed using ascorbic acid.

26. The method of claim 23, wherein said pattern of reduced graphene oxide comprises one continuous layer.

27. The method of claim 23, wherein said biomimetic matrix has a two-dimensional structure.

28. The method of claim 23, wherein said obtaining step comprises the step of electrospinning nonwoven fibers to form said biomimetic matrix.

29-30. (canceled)

31. The method of claim 22, wherein said cationic polyelectrolyte comprises branched polyethylenimine.

32. The method of claim 22, wherein multiple layers of said pattern of reduced graphene oxide are formed.

33-34. (canceled)

35. The method of claim 22, wherein said obtaining step comprises the step of printing said biomimetic matrix via near-field electrostatic printing.

36. The method of claim 22, wherein said biomimetic matrix has a three-dimensional structure.

37. The method of claim 35, wherein said biomimetic matrix is printed with a forty-five degree overlay angle.

38. The method of claim 35, wherein said biomimetic matrix is printed with a sixty degree overlay angle.

39. The method of claim 35, wherein said biomimetic matrix is printed with a seventy-five degree overlay angle.

40. The method of claim 35, wherein said biomimetic matrix is printed with a ninety degree overlay angle.

41. The method of claim 35, wherein said biomimetic matrix is printed with fiber diameters ranging from about 15 picometers to about 150 picometers.

42. The method of claim 35, wherein said biomimetic matrix is printed with a spider web microfiber pattern.

43. The method of claim 35, wherein said biomimetic matrix is printed with a cylindrical microfiber pattern.

44. The method of claim 35, wherein said printing step is conducted using a layer by layer printing technique.

45. The method of claim 35, wherein said electrically-conductive scaffold comprises microfibers having microfiber diameters tailored to induce neural network formation along said microfibers under electrical stimulation.

46. The method of claim 35, wherein said electrically-conductive scaffold comprises a pattern of microfibers tailored to induce neural network formation along said microfibers of said pattern under electrical stimulation.

Description

BRIEF DESCRIPTION OF FIGURES

[0022] For a more complete understanding of the present disclosure, reference is made to the following drawings, in which:

[0023] FIG. 1 is a schematic diagram of an exemplary system for making scaffolds in accordance with an embodiment of the present invention;

[0024] FIG. 2 is a flow diagram illustrating an exemplary method of making scaffolds in accordance with an embodiment of the present invention;

[0025] FIG. 3 is a schematic diagram of a 2D inkjet printing-based fabrication process for scaffolds with conductive micropatterns;

[0026] FIG. 4 is a series of stereomicroscope images and scanning Electron Microscope images of scaffolds made in accordance with embodiments of the present invention;

[0027] FIG. 5 is a collection of stereomicroscope images of different printing patterns on scaffolds made in accordance with embodiments of the present invention;

[0028] FIG. 6A is a collection of stereomicroscopic images of graphene oxide printed stripes with different widths, before reduction;

[0029] FIG. 6B is a collection of stereomicroscopic and SEM images of graphene oxide-printed stripes with different widths, after reduction;

[0030] FIG. 6C is a collection of stereomicroscopic images of graphene oxide-printed patterns, before reduction;

[0031] FIG. 6D is a collection of stereomicroscopic images of graphene oxide-printed patterns, after reduction;

[0032] FIG. 7A is a bar graph displaying the measured electrical conductivity of rGO stripes with various printed layers in accordance with an embodiment of the present invention;

[0033] FIG. 7B is a pair of macroscopic views of a lit-up LED bulb between two 4-layer rGO stripes;

[0034] FIG. 8A is a bar graph displaying the measured stress-strain curve of exemplary materials that could be utilized as substrate materials for inkjet printing in accordance with an embodiment of the present invention;

[0035] FIG. 8B shows the recovery testing results for the substrate materials of FIG. 8A;

[0036] FIGS. 9A-9C are schematic illustrations of conditions for different rGO printed patterns, which figures correspond to FIGS. 9D-9G, FIGS. 9H-9K, and FIGS. 9L-9O, respectively;

[0037] FIGS. 9D-9G are a series of epifluorescent images of PC12 cells cultured on nanofiber matrices with and without electrical stimulation after culturing for 7 and 14 days, under the conditions represented in FIG. 9A;

[0038] FIGS. 9H-9K are a series of epifluorescent images of PC12 cells seeded on inkjet-printed stripes on nanofiber matrices after 14 days of culturing under different electrical stimulation conditions;

[0039] FIGS. 9L-9O are a series of epifluorescent images of PC12 cells seeded on the inkjet-printed circle patterns on nanofiber matrices after 14 days of culturing under different electrical stimulation conditions;

[0040] FIG. 10 is a series of schematic illustrations showing the production of 3D conductive microfiber scaffolds based on near-field electrostatic printed constructs;

[0041] FIG. 11 is a series of images of PLCL microfibers coated with graphene oxide and those coated with reduced graphene oxide, taken with a stereomicroscope (FIG. 11A) or scanning electron microscope (FIGS. 11B-11D) with various magnifications;

[0042] FIG. 12A is an illustration/morphology of microfibers with different angles, in accordance with an embodiment of the present invention;

[0043] FIG. 12B is an illustration/morphology of microfibers in a spider web-like structure, in accordance with an embodiment of the present invention;

[0044] FIG. 12C is an illustration/morphology of microfibers in a large volume cylindrical construct structure, in accordance with an embodiment of the present invention;

[0045] FIG. 12D is an illustration/morphology of microfibers with different diameters, in accordance with an embodiment of the present invention;

[0046] FIG. 13A is a schematic illustration of an electrical stimulation apparatus used in connection with an embodiment of the present invention;

[0047] FIGS. 13B and 13C are color maps of applied electrical stimulation in accordance with an embodiment of the present invention;

[0048] FIGS. 14A-14J are a series of immunostaining images and a corresponding intensity plot of PC12 cells seeded onto graphene oxide/reduced graphene oxide coated PLCL microfibers with different levels of electrical stimulation, in accordance with an embodiment of the present invention;

[0049] FIG. 14K is a corresponding intensity plot of the images of FIGS. 14A-14J; and

[0050] FIGS. 15A-1 to 15A-6, FIGS. 15B-1 to 15B-10, FIGS. 15C-1 to 15C-4, and a fourth group that includes FIGS. 6D-1, 6D-2, 6D-4, 6D-5 and 6D-7, are each a series of immunostaining images for neurites after 21 days culturing of PC12 cells seeded onto graphene oxide/reduced graphene oxide coated PLCL microfibers with various patterns, in accordance with embodiments of the present invention, FIG. 15D-3 being a cylindrical construct corresponding to FIGS. 6D-1 and 6D-2, and FIG. 15D-6 being a cylindrical construct corresponding to FIGS. 6D-4, 6D-5, and 6D-7.

DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS

[0051] Reference will now be made to several embodiments of the present invention(s), examples of which are illustrated in the accompanying figures. Wherever practicable similar or like reference numbers may be used in the figures and may indicate similar or like functionality. The figures depict embodiments of the present invention for purposes of illustration only. One skilled in the art will readily recognize from the following description that alternative embodiments of the structures and methods illustrated herein may be employed without departing from the principles of the invention described herein.

[0052] All terms defined herein should be afforded their broadest possible interpretation, including any implied meanings as dictated by a reading of the specification as well as any words that a person having skill in the art and/or a dictionary, treatise, or similar authority would assign thereto. The terms, “for example”, “e.g.”, “optionally”, as used herein, are intended to be used to introduce non-limiting examples. The phrases “in one embodiment” and “in some embodiments” as used herein do not necessarily refer to the same embodiment(s), though they may. Furthermore, the phrases “in another embodiment” and “in some other embodiments” as used herein do not necessarily refer to a different embodiment, although they may. Thus, as described below, various embodiments of the invention may be readily combined, without departing from the scope or spirit of the invention.

[0053] In addition, as used herein, the term “or” is an inclusive “or” operator, and is equivalent to the term “and/or,” unless the context clearly dictates otherwise. The term “based on” is not exclusive and allows for being based on additional factors not described, unless the context clearly dictates otherwise. In addition, throughout the specification, the meaning of “a,” “an,” and “the” include plural references. The meaning of “in” includes “in” and “on.” In addition, the terms “comprises” and “comprising” when used herein specify that certain features are present in that embodiment; however, this phrase should not be interpreted to preclude the presence or addition of additional steps, operations, features, components, and/or groups thereof.

[0054] Conductive scaffolds in accordance with the present invention may fabricated by depositing soluble graphene oxide on substrate surfaces, as well as by coating onto the 3D microfiber scaffolds layer-by-layer and then reducing into conductive reduced graphene oxide. With highly controllable deposition or coating to any arbitrary surface, the conductive scaffolds of the present invention can achieve various configurations, with potential for mass production.

[0055] In general, to obtain conductive scaffolds, a substrate made from synthetic materials such as PLCL, natural materials such as collagen, or hybrid materials such as PLCL/collagen is desirable, as the scaffold will need to take forms such as sponges, fibers, meshes, membranes, spheres, etc. On top of such a substrate, graphene oxide (GO) will be deposited from an aqueous solution in a layer-by-layer manner by alternating it with cationic polyelectrolyte, such as branched polyethylenimine (BPEI), in order to achieve different thicknesses and strong bonding. Upon reaching the desired thickness, the substrate with deposited GO will be reduced to reduced graphene oxide (rGO) with reductants such as ascorbic acid. Upon completion of the reduction, the scaffolds will be conductive for use in electrical stimulation.

[0056] Unique conductive features of the scaffolds can be afforded depending on the manufacturing technique. For example, applied inkjet printing can be used to achieve 2D spatial conductive patterns on biomimetic fibrous matrices. Specifically, a synthetic polymer of PLCL, which has demonstrated potential utility in nerve tissue engineering, may be electrospun into nonwoven nanofiber matrices to mimic the native tissue extracellular matrix (ECM). Then GO solution can be loaded into a printer cartridge as “ink” and be printed onto the fibrous matrices, which essentially serve as “paper.” During printing, the GO solution is precisely deposited onto the matrix surface to generate a continuous layer of a GO pattern following a pre-designed pattern. After reducing the printed GO, a pattern of rGO can be formed on the fiber matrix. The obtained conductive patterns exhibited ability to promote the neurite ingrowth along the rGO printed area under electrical stimulation.

[0057] In another embodiment, 3D conductive scaffolds can be deposited on printed microfiber structures using near-field electrostatic printing (NFEP). An appropriate material is printed into different microfiber templates using NFEP. Various microfiber patterns may be designed accordingly, including those with different fiber overlay angles (45°, 60°, 75°, 90°), different fiber diameters (e.g., ranging from 15 μm to 150 μm) and complex structures (e.g., “spider web” or “cylinder” structures). Once the microfiber structure is fabricated, GO solution may be coated onto PLCL microfibers via layer-by-layer technique and in situ reduced into rGO. The obtained conductive scaffolds showed capability of inducing the neural network formation along the conductive microfibers under electrical stimulation. Both pattern and fiber diameter could regulate neural cell differentiation and neurite outgrowth under electrical stimulation.

[0058] FIG. 1 illustrates schematically a system for producing scaffolds in accordance with the present invention, while FIG. 2 outlines the process for same.

[0059] Prototypes of conductive micropatterns on PLCL nanofiber matrices and conductive 3D PLCL microfiber scaffolds have been fabricated as described above. The scaffolds were then characterized by a scanning electron microscope (SEM) and optical microscope to observe the morphology and microstructure of the obtained scaffolds.

[0060] Prototypes of conductive micropatterns on PLCL nanofiber matrices are depicted in FIG. 3.

[0061] FIG. 4 shows stereomicroscope and SEM images of various resultant patterns, resulting from varying parameters on such scaffolds, with a scale bar of 200 μm. FIG. 5 shows further stereomicroscope images of resulting patterns, with a scale bar of 200 μm. FIG. 6 shows stereomicroscopic and SEM images of printed graphene oxide stripes of various widths, and complex shapes of patterns before and after reduction, with a scale bar of 200 μm.

[0062] FIG. 10 illustrates schematically the production of 3D conductive microfiber scaffolds manufactured with near-field electrostatic printed constructs. FIG. 11 shows the morphology of graphene oxide/reduced graphene oxide coated PLCL microfibers with varying coating layers, via stereomicroscopy (FIG. 11A; scale bar: 150 μm) and scanning electron microscopy at various magnifications, wherein the depicted scale bars are 500 μm, 1 μm and 12 μm, respectively, for FIGS. 11B-11D.

[0063] FIG. 12 is an illustration of morphology of various printing patterns of microfibers made in accordance with an embodiment of the present invention, showing microfibers with different angles (FIG. 12A), spider web-like structures (FIG. 12B), large volume cylindrical constructs (FIG. 12C) and with different diameters (FIG. 12D), wherein all scale bars represent 150 μm.

[0064] The coating of GO/rGO layers on the surface of arbitrary substrates may yield conductive scaffolds exhibiting exceptional unique topographical features and excellent electrical conductivity. In view of dominant efforts to guide neural network formation via scaffold topography on a planar substrate such as silicon wafer, the current invention develops unique conductive scaffolds for neural network formation under electrical stimulation and enables the formation of 3D neural networks similar to the native tissue network. More specifically, hierarchical neural network structure with biological functions may be established.

[0065] The electric conductivity of the prepared scaffolds was also measured, through a customized resistance measurement apparatus. Quantitative measurements of electrical conductivity for the different rGO printing layers are shown in FIG. 7A for inkjet-printed scaffolds. FIG. 7B shows macroscopic views of lighting up an LED lamp with 4 layers of rGO printing.

[0066] The mechanical properties of PLCL (50:50) as an inkjet printing substrate was also measured through comparison with other substrate materials (i.e., polylactic acid (PLA), polycaprolactone (PCL), and PLCL (80:20)). FIG. 8A shows the stress-strain curve for the materials. The quantitative measurements of recovery testing for the substrate materials are illustrated in FIG. 8B.

[0067] FIG. 13A is a schematic illustration of an electrical apparatus used for testing, and FIGS. 13B and 13C are simulations of electrical stimulation applied for in vitro study, showing, specifically, COMSOL simulation results for the electrical potential changes for NFEP-printed microfiber scaffolds.

[0068] Both types of scaffolds were used to culture neural cells (PC12 cells) for a designated period under the electrical stimulation and then immunofluorescently stained with biomarkers for evaluating neurite outgrowth and neural network formation.

[0069] FIG. 9 shows immunostaining for neurites after culturing PC12 cells seeded on the inkjet-printed nanofiber matrices with and without electrical stimulation for 7 and 14 days. Epifluorescent images were taken after immunofluorescently staining for nuclei (DAPI, blue), Beta tubulin III (green) and Phalloidin (red). The given scale bars correspond to 100 μm.

[0070] FIG. 14 illustrates immunostaining for neurites after 21 days culturing of PC12 cells seeded onto GO/rGO coated PLCL microfibers made with NFEP, given different electrical stimulation levels, and wherein the given scale bars correspond to 150 μm. FIG. 15 shows immunostaining for neurites after 21 days culturing of PC12 cells seeded onto GO/rGO coated NFEP-printed PLCL microfibers with various patterns. FIGS. 15A-1 to 15A-6 show epifluorescent images for microfibers with different diameters; FIGS. 15B-1 to 15B-10 show epifluorescent images for microfibers with different angles; FIGS. 15C-1 to 15C-4 show Confocal Laser Scanning Microscope images for spiderweb-like structures; and FIGS. 15D-1 to 15D-7 show Confocal Laser Scanning Microscope images for large volume cylindrical constructs (see FIGS. 15D-3 and 15D-6). Epifluorescent images and Confocal Laser Scanning Microscope images were taken after immunofluorescently staining for nuclei (DAPI, blue), Beta tubulin III (green) and Phalloidin (red). The given scale bars represent 150 μm.

[0071] Details are provided below for the fabrication of 2D conductive micropatterns on biomimetic nanofibrous matrices using inkjet printing and 3D conductive microfiber scaffolds using NFEP. Both demonstrations can be extended to other substrates with broad applicability.

Example 1—Formation of 2D Conductive Micropatterns Using Inkjet Printing

[0072] PLCL solution at 5-12% (w/v) was prepared via thorough mixing of PLCL in 1, 1, 1, 3, 3, 3-Hexafluoro-2-propanol (HFIP) for at least 12 hours prior to electrospinning. For the fabrication of nanofibrous matrices using electrospinning, the solution was transferred to a syringe equipped with a tip-blunt nozzle. The solution was pumped through the nozzle at a steady flow rate of 10 μL/min. To pull the solution into nanofibers, a high voltage of 10-20 kV was applied between the nozzle and a grounded collector, with a distance of 15 cm. The collected nanofiber matrices were vacuum dried to remove any trace amount of HFIP for further use. To form conductive micropatterns on the nanofiber matrices, aqueous solutions of graphene oxide (1-5 mg/mL) were used as the ink and loaded into the cartridge of a specialized printer (e.g., FUJIFILM Dimatix Materials Printer DMP-2800). Following pre-drawn AutoCAD patterns, graphene oxide ink was printed onto the nanofiber matrices. Optimization of the printing parameters was carried out by varying the drop distance (DD) and printing layers with various pattern combinations. After printing, the printed scaffolds were transferred to a container containing ascorbic acid solution (e.g., 10-30 mM) and reduced for 2-5 hours. After reduction, all scaffolds were rinsed with deionized water and air-dried. The reduced graphene oxide (rGO) micropatterns enabled the formation of neural networks upon the culture of neural cells on the surface with provided electrical stimulation (100-150 mV/cm). Such matrices can be further processed into tubes or other 3D structures.

Example 2—Formation of 3D Conductive Microfiber Scaffolds Using NFEP

[0073] The preparation process may be divided into three steps: NFEP of a PLCL microfiber construct, L-b-L GO-coating onto microfibers, and reduction of GO to rGO.

[0074] First, 3D microfiber constructs of PLCL are fabricated from PLCL solution (80-120 mg/mL in HFIP) using near-field electrostatic printing. The PLCL solution may be loaded into a syringe with a blunt tip nozzle and pumped through the nozzle via a syringe pump at a rate of 0.4-1 μL/min. An electric field of 1-3 kV may be applied between the nozzle and the collection surface. Close collection distance (0.5-3 mm) allows direct deposition of the stretched jet of PLCL solution onto the grounded collection surface to form 3D structures with desirable patterns upon HFIP evaporation. To form 3D tubular constructs of PLCL microfibers, the collection surface is replaced with a rotating stainless-steel rod. The printed patterns may be designed and programmed in g-code. By modulating the collection distance (H), voltage (V), nozzle diameter and solution concentration, the diameter of printed PLCL microfibers can be tuned in the range of 15-150 μm.

[0075] The obtained microfiber constructs are then coated with GO, with the help of BPEI by alternating the incubation solution between BPEI (e.g., at 1-5 mg/mL in 0.1 M phosphate buffer) and GO solution (e.g., 1-5 mg/mL). After reaching the desirable layers of GO coating, the constructs may be rinsed with 0.1 M phosphate buffer and then transferred to a container of ascorbic acid solution (e.g., 10-30 mM) and reduced for 2-5 hours. After reduction, all scaffolds may be rinsed with deionized water and air-dried. The reduced rGO microfiber constructs enable the formation of 3D neural networks upon the culture of neural cells within the scaffolds under electrical stimulation (100-150 mV/cm).

[0076] It will be understood that the embodiments described hereinabove, are merely exemplary and that a person skilled in the art may make many variations and modifications without departing from the spirit and scope of the present invention. All such variations and modifications are intended to be included within the scope of the invention.