CN111944750B - Three-dimensional annular cell scaffold with radio stimulation response and preparation method and application thereof - Google Patents
Three-dimensional annular cell scaffold with radio stimulation response and preparation method and application thereof Download PDFInfo
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
- C12N5/0662—Stem cells
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/0062—General methods for three-dimensional culture
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- C23—COATING METALLIC MATERIAL; COATING MATERIAL WITH METALLIC MATERIAL; CHEMICAL SURFACE TREATMENT; DIFFUSION TREATMENT OF METALLIC MATERIAL; COATING BY VACUUM EVAPORATION, BY SPUTTERING, BY ION IMPLANTATION OR BY CHEMICAL VAPOUR DEPOSITION, IN GENERAL; INHIBITING CORROSION OF METALLIC MATERIAL OR INCRUSTATION IN GENERAL
- C23C—COATING METALLIC MATERIAL; COATING MATERIAL WITH METALLIC MATERIAL; SURFACE TREATMENT OF METALLIC MATERIAL BY DIFFUSION INTO THE SURFACE, BY CHEMICAL CONVERSION OR SUBSTITUTION; COATING BY VACUUM EVAPORATION, BY SPUTTERING, BY ION IMPLANTATION OR BY CHEMICAL VAPOUR DEPOSITION, IN GENERAL
- C23C16/00—Chemical coating by decomposition of gaseous compounds, without leaving reaction products of surface material in the coating, i.e. chemical vapour deposition [CVD] processes
- C23C16/01—Chemical coating by decomposition of gaseous compounds, without leaving reaction products of surface material in the coating, i.e. chemical vapour deposition [CVD] processes on temporary substrates, e.g. substrates subsequently removed by etching
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- C23—COATING METALLIC MATERIAL; COATING MATERIAL WITH METALLIC MATERIAL; CHEMICAL SURFACE TREATMENT; DIFFUSION TREATMENT OF METALLIC MATERIAL; COATING BY VACUUM EVAPORATION, BY SPUTTERING, BY ION IMPLANTATION OR BY CHEMICAL VAPOUR DEPOSITION, IN GENERAL; INHIBITING CORROSION OF METALLIC MATERIAL OR INCRUSTATION IN GENERAL
- C23C—COATING METALLIC MATERIAL; COATING MATERIAL WITH METALLIC MATERIAL; SURFACE TREATMENT OF METALLIC MATERIAL BY DIFFUSION INTO THE SURFACE, BY CHEMICAL CONVERSION OR SUBSTITUTION; COATING BY VACUUM EVAPORATION, BY SPUTTERING, BY ION IMPLANTATION OR BY CHEMICAL VAPOUR DEPOSITION, IN GENERAL
- C23C16/00—Chemical coating by decomposition of gaseous compounds, without leaving reaction products of surface material in the coating, i.e. chemical vapour deposition [CVD] processes
- C23C16/22—Chemical coating by decomposition of gaseous compounds, without leaving reaction products of surface material in the coating, i.e. chemical vapour deposition [CVD] processes characterised by the deposition of inorganic material, other than metallic material
- C23C16/26—Deposition of carbon only
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- C12N2529/00—Culture process characterised by the use of electromagnetic stimulation
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Abstract
The invention discloses a three-dimensional annular cell scaffold with radio stimulation response, and a preparation method and application thereof. The preparation method comprises the following steps: growing graphene on a copper/nickel template by adopting a chemical vapor deposition method to prepare a three-dimensional annular cell bracket; or conducting material and fiber are subjected to blending treatment to form conducting fiber, and then a template method is adopted for treatment to prepare the three-dimensional annular cell scaffold; or printing the conductive hydrogel and/or the conductive aerogel by adopting a 3D printing method to prepare the three-dimensional annular cell scaffold. The three-dimensional annular cell scaffold prepared by the invention has the advantages of accurate and controllable size and good conductivity; meanwhile, the three-dimensional annular cell scaffold inoculated with the mesenchymal stem cells is simple and convenient by utilizing radio stimulation, the trouble of wire connection is solved, the problems of low flow and survival rate of the mesenchymal stem cells in tissues and cell transplantation damage are reduced, and the cell behavior is controllable.
Description
Technical Field
The invention belongs to the technical field of new nano materials, and particularly relates to a three-dimensional annular cell scaffold with radio stimulation response, and a preparation method and application thereof.
Background
Mesenchymal Stem Cells (MSC) are important members of stem cell families, are one of pluripotent stem cells, and have the characteristics of self replication, multidirectional differentiation potential, stem cell implantation promotion, hematopoietic support, immune regulation and the like; MSC can differentiate into various tissue cells such as fat, bone, cartilage, muscle, tendon, ligament, nerve, liver, cardiac muscle, endothelial and the like under specific induction conditions in vivo or in vitro, has multidirectional differentiation potential after continuous subculture and cryopreservation, and can be used as ideal seed cells for repairing tissue and organ injury caused by aging and pathological changes.
Graphene is a carbon nanomaterial composed of a single layer or a few layers of carbon atoms, has excellent physicochemical properties, such as extremely high electron mobility, adjustable optical properties, high mechanical strength and good heat and electrical conductivity, and has received wide attention in the fields of materials, physics and chemistry. The graphene prepared by the template method has fewer defect stacks, and in the biological application process, the graphene is found to have a porous structure, a larger specific surface area and a unique surface topology structure, so that the cell growth microenvironment can be better simulated. When the graphene scaffold is used for cell culture, the graphene scaffold has good biocompatibility and can obviously promote directional differentiation of MSC. In addition, graphene is used as a conductive material, and cell behaviors can be regulated and controlled in an electrical stimulation mode.
The hydrogel is a water-swellable crosslinked polymer network generated by simple reaction of one or more monomers, can be formed by certain chemical crosslinking or physical crosslinking of water-soluble or hydrophilic polymers, combines the redox conversion capability of the conductive polymer with the rapid ion mobility and biocompatibility of the hydrogel, can be produced into nano conductive hydrogel templates of various sizes and combined with bioactive molecules, simulates extracellular matrixes and is used for cell culture. In addition, the conductive hydrogel is used as a conductive material, and can regulate and control the cell behavior in an electrical stimulation mode.
The conductive fiber refers to chemical fiber or metal fiber, carbon fiber, conductive polymer fiber, etc. spun by mixing conductive medium into polymer, and is produced into nanometer conductive fiber template with various sizes and bioactive molecules through mixing, evaporating, electroplating, composite spinning, etc. to simulate extracellular matrix for cell culture. In addition, the conductive fiber is used as a conductive material, and can regulate and control the cell behavior in an electrical stimulation mode.
Aerogels, when the gel is freed of most of the solvent, make the liquid content in the gel much less than the solid content, or the medium filling the space network of the gel is a gas, the appearance is solid. The redox conversion capability of the conductive polymer is combined with the rapid ion mobility and biocompatibility of the aerogel, so that nano conductive aerogel templates with various sizes and bioactive molecules can be produced, and extracellular matrixes can be simulated for cell culture. In addition, the conductive aerogel is used as a conductive material, and can regulate and control the cell behavior in an electrical stimulation mode.
The graphene structure with precisely controlled shape, size and the like can be prepared by combining micro-nano processing technology such as photoetching, electroplating and the like with CVD technology, but the application of electric stimulation to the graphene structure still has the obstacle: how to make good connection with the external lead. The graphene has higher rigidity, when connecting harder platinum wires, the structural integrity of the contact part of the graphene and the platinum wires is difficult to maintain, gaps exist in the connection of the graphene and the platinum wires, when high-conductivity liquid such as silver paste is used for assisting in connection, the penetration range of the liquid such as silver paste is difficult to control, and especially whether high-conductivity auxiliary materials such as silver paste additionally influence cells is also very required. In addition, there are also obstacles to the connection of conductive hydrogels, conductive fibers, conductive aerogels, etc. to external leads. On the other hand, in the case of mesenchymal stem cell transplantation, the wire connection means that both the wire and the power source need to be fixed in or on the body surface of the animal, which makes the transplantation of the stent and the anti-infection operation difficult.
Disclosure of Invention
The invention mainly aims to provide a three-dimensional annular cell scaffold with radio stimulation response, a preparation method and application thereof, so as to overcome the defects of the prior art.
In order to achieve the purpose of the invention, the technical scheme adopted by the invention comprises the following steps:
the embodiment of the invention provides a preparation method of a three-dimensional annular cell scaffold with radio stimulation response, which comprises the following steps:
growing graphene on a copper/nickel template by adopting a chemical vapor deposition method to prepare a three-dimensional annular cell bracket;
or conducting material and fiber are subjected to blending treatment to form conducting fiber, and then a template method is adopted for treatment to prepare the three-dimensional annular cell scaffold;
or printing the conductive hydrogel and/or the conductive aerogel by adopting a 3D printing method to prepare the three-dimensional annular cell scaffold.
The embodiment of the invention also provides the three-dimensional annular cell scaffold prepared by the method.
The embodiment of the invention also provides the application of the three-dimensional annular cell scaffold in cell culture.
The embodiment of the invention also provides a culture method of the mesenchymal stem cells, which comprises the following steps:
providing the three-dimensional annular cell scaffold;
and inoculating the mesenchymal stem cells to the three-dimensional annular cell scaffold, and then performing radio stimulation on the three-dimensional annular cell scaffold inoculated with the mesenchymal stem cells by using a primary coil with an electric signal, thereby regulating and controlling proliferation and differentiation of the mesenchymal stem cells.
Compared with the prior art, the invention has the beneficial effects that:
(1) According to the preparation method, copper/nickel is used as a template, and the graphene three-dimensional annular cell bracket with a controllable structure is prepared by using a chemical vapor deposition method, so that the defects are few, and the quality is good; preparing a conductive hydrogel and conductive aerogel three-dimensional annular cell bracket with controllable structure by using a 3D printer; the three-dimensional annular cell scaffold is prepared by utilizing the conductive fibers, and the size of the three-dimensional annular cell scaffold prepared by the method is accurate and controllable, and the conductivity is good;
(2) The invention uses radio to stimulate cells, is simple and convenient, reduces the problems caused by wire connection, reduces the problems of low flow and survival rate of mesenchymal stem cells in tissues, and reduces cell transplantation damage; the invention induces the directional differentiation of the mesenchymal stem cells by adjusting various physical and chemical characteristics of the three-dimensional annular cell scaffold; the invention combines the advantages of radio stimulation and structure controllable three-dimensional annular cell bracket to regulate and control cell behavior; the three-dimensional annular cell scaffold is combined with sustained and controlled release, so that the nutritional factors are released, and the cell behaviors are regulated; the cell culture method provided by the invention can stimulate the expression of exosomes and regulate and control the cell behaviors; the cell culture method provided by the invention has anti-inflammatory capability on external injury, and in addition, the characteristics of conductivity, aperture, porosity, specific surface area and the like of the bracket can be adjusted by changing the proportion of materials and parameters in the preparation process and the size of the annular bracket.
Drawings
In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings that are required in the embodiments or the description of the prior art will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments described in the present application, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is an X-ray energy spectrum of a three-dimensional graphene toroidal cell scaffold prepared in example 1 of the present invention;
FIG. 2 is a Raman spectrum of a three-dimensional graphene toroidal cell scaffold prepared in example 1 of the present invention;
FIG. 3 is a schematic diagram showing the cell culture using radio stimulation in example 1 of the present invention;
FIGS. 4a-4b are electrical signals of induced electromotive force generated by the three-dimensional toroidal cell-scaffold in example 1 of the present invention, respectively.
FIGS. 5a-5c are graphs showing the effect of radio stimulation on cell differentiation after seven days;
FIGS. 6a-6b are scanning electron microscope images of a three-dimensional hydrogel annular cell scaffold prepared in example 2 of the present invention;
FIG. 7 is a chart showing pore size statistics of a three-dimensional hydrogel annular cell scaffold prepared in example 2 of the present invention;
FIGS. 8a-8c are graphs of the biocompatibility of a three-dimensional hydrogel annular cell scaffold prepared in example 2 of the present invention.
Detailed Description
In view of the shortcomings of the prior art, the inventor of the present application has long studied and put forward a great deal of practice, and the technical solution of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are some, but not all, embodiments of the present invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
One aspect of an embodiment of the present invention provides a method for preparing a three-dimensional annular cell scaffold for radio stimulation response, comprising:
growing graphene on a copper/nickel template by adopting a chemical vapor deposition method to prepare a three-dimensional annular cell bracket;
or conducting material and fiber are subjected to blending treatment to form conducting fiber, and then a template method is adopted for treatment to prepare the three-dimensional annular cell scaffold;
or printing the conductive hydrogel and/or the conductive aerogel by adopting a 3D printing method to prepare the three-dimensional annular cell scaffold.
In some more specific embodiments, the method of making comprises: at least adopting any micro-nano processing means of photoetching, plasma etching and electroplating to prepare a copper/nickel template with a controllable structure, then adopting a chemical vapor deposition method to grow graphene on the copper/nickel template, and then carrying out corrosion, cleaning and drying treatment to prepare the three-dimensional annular cell bracket.
In some more specific embodiments, the chemical vapor deposition process comprises:
providing a three-dimensional annular copper/nickel template as a catalyst and placing the three-dimensional annular copper/nickel template in a reaction chamber of chemical vapor growth equipment;
introducing hydrogen and argon into the reaction chamber, and carrying out annealing treatment for 10-15min at 950 ℃;
and introducing mixed gas of hydrogen, argon and methane into the reaction chamber, growing a graphene layer on the surface of the three-dimensional annular copper/nickel template by a chemical vapor deposition method, and then carrying out etching treatment to obtain the three-dimensional annular cell support.
Further, the preparation method further comprises the following steps: and cleaning the three-dimensional annular copper/nickel template, drying and then placing the three-dimensional annular copper/nickel template in the reaction chamber.
Further, the preparation method comprises the following steps: and heating the three-dimensional annular copper/nickel template in the reaction chamber to 950 ℃ under the low-flow hydrogen and high-flow argon atmosphere, and then annealing for 10-15min under the high-flow hydrogen and low-flow argon atmosphere.
Further, the macroscopic inner diameter of the three-dimensional annular cell scaffold is 4-9mm, and the outer diameter of the three-dimensional annular cell scaffold is 14-20mm.
In some more specific embodiments, the method of preparing a three-dimensional annular cell scaffold by chemical vapor deposition comprises:
and growing the three-dimensional annular graphene in a horizontal tubular CVD furnace by taking the three-dimensional annular copper/nickel template with controllable inner and outer diameters as a catalyst. Firstly, sequentially placing copper/nickel in absolute ethyl alcohol and deionized water for ultrasonic treatment for 15min to remove surface impurities, sequentially immersing in 0.1mol/L dilute hydrochloric acid for 2min and deionized water for 5min for further cleaning, and drying with nitrogen. At low flow rate H 2 And heating the template to 950 ℃ under high flow rate Ar gas, at high flow rate H 2 And low flow Ar gas for 10min, and then exposed to low flow H 2 High flow Ar and CH 4 The next 5min, finally the CVD system was cooled to room temperature. And placing the template growing with the graphene in ferric chloride corrosive liquid until the template is corroded completely. And sequentially placing the obtained sample into 1mol/L, 0.1mol/L, 0.01mol/L dilute hydrochloric acid and deionized water for cleaning, and then gradually dehydrating and freeze-drying in graded ethanol to prepare the three-dimensional annular cell scaffold.
In some more specific embodiments, the method of making comprises: conducting materials and fibers are subjected to blending treatment to form conducting fibers, and then the conducting fibers are processed through a template to obtain the three-dimensional annular cell scaffold;
further, the macroscopic inner diameter of the three-dimensional annular cell scaffold is 4-9mm, and the outer diameter of the three-dimensional annular cell scaffold is 14-20mm.
Further, the conductive substance includes any one or a combination of two or more of metal, carbon black, conductive polymer, and metal compound, and is not limited thereto.
In some more specific embodiments, the blending process comprises: the conductive substrate and the fiber are sliced, and the conductive fiber is prepared through a pulse conveyer, a wet slice large bin, a pulse conveyer, a pre-crystallizer (the pre-crystallization temperature is determined according to the fiber), a drying tower (air temperature and time), an extruder, a melt distributing pipe, a spinning box, a metering pump, a composite spinning component, side blowing, bundling oiling, winding and chemical fiber, and the blending time is determined according to the material.
In some more specific embodiments, the conductive hydrogel and/or conductive aerogel comprises inorganic and/or conductive polymers therein.
Further, the inorganic substance includes any one or a combination of two or more of graphene, graphite, carbon fiber, carbon nanotube, and metal particle, and is not limited thereto.
Further, the conductive polymer includes any one or a combination of more than two of polypyrrole, polyethylene terephthalate, polylactic acid, nanocellulose, dextran, carbon nanotubes, polyacrylamide and mucopolysaccharide, and is not limited thereto.
Further, the conductive polymer includes polyaniline and any one or a combination of more than two of polypropylene, polycaprolactone, polyvinylphenol and polyacrylamide, and is not limited thereto.
Further, the three-dimensional annular cell scaffold obtained by 3D printing has a macroscopic internal diameter of 4-9mm, an external diameter of 14-20mm and a microscopic aperture size of 2-14 mu m.
In some more specific embodiments, preparing a three-dimensional annular cell scaffold using a 3D printing method comprises: the 3D printing is one kind of fast forming technology, and is one kind of technology of constructing object with powdered metal, plastic or other adhesive material based on digital model file in layer-by-layer printing mode. The steps are as follows: 1. modeling: manually modeling, and designing a three-dimensional annular structure with controllable inner and outer diameters by using 3D-MAX, Z-Brush and other software; 3D scanning, namely scanning the three-dimensional annular support template by a 3D scanner to generate a digital file. 2. Slicing: the built 3D digital model is converted into a walking path which can be identified by a 3D printer and the extrusion amount of consumable materials, the model is firstly loaded into software, a model slice is clicked, and after the model slice is finished, a file is sent to the 3D printer. 3. Printing: find file, click to start printing. The device was placed on a 3D printer and the central container of the device was filled with the required amount of hydrogel.
Further, the hydrogel includes graphene and polysaccharide.
In some more specific embodiments, the method of preparing a three-dimensional annular cell scaffold comprises:
manufacturing a copper/nickel template with a controllable structure by micro-nano processing means such as photoetching, plasma etching, electroplating and the like, growing graphene on the template by a chemical vapor deposition method, and preparing the three-dimensional annular cell bracket by corrosion, cleaning and airing;
or preparing inorganic matter-added conductive hydrogel and/or conductive polymer-based conductive hydrogel by adding inorganic matter and/or conductive polymer, and processing into a three-dimensional annular cell bracket with a certain size by a 3D printer;
or, blending conductive substances (metal, carbon black, conductive polymer and metal compound) with common fibers to prepare conductive fibers, and processing the conductive fibers into a three-dimensional annular cell bracket with a certain size through a template;
or preparing inorganic substance added conductive aerogel and/or conductive polymer based conductive gel by adding inorganic substance and/or conductive polymer, and processing into three-dimensional annular cell scaffold with certain size by 3D printer.
In another aspect, the embodiment of the invention also provides the three-dimensional annular cell scaffold prepared by the method.
Another aspect of embodiments of the present invention also provides the use of the three-dimensional toroidal cell scaffold described above in culturing cells.
Further, the cells are mesenchymal stem cells, and are not limited thereto.
Another aspect of the embodiments of the present invention also provides a method for culturing mesenchymal stem cells, comprising:
providing the three-dimensional annular cell scaffold;
and inoculating the mesenchymal stem cells to the three-dimensional annular cell scaffold, and then performing radio stimulation on the three-dimensional annular cell scaffold inoculated with the mesenchymal stem cells by using a primary coil with an electric signal, thereby regulating and controlling proliferation and differentiation of the mesenchymal stem cells.
Further, the electrical signal is a sinusoidal alternating current signal: the frequency is 20kHz, the current is 2A, and the stimulation duration is as follows: strings are 1s long, strings are spaced 2s apart, 1h each day, and one period is 7 days.
Further, the method further comprises: and (3) inoculating the mesenchymal stem cells to the three-dimensional annular cell scaffold, and then performing wireless electric stimulation on the three-dimensional annular cell scaffold inoculated with the mesenchymal stem cells by using a primary coil with an electric signal to promote the expression of exosomes, so as to promote any one of cell migration, differentiation, antigen presentation or organism immune response.
Further, the three-dimensional annular cell scaffold also comprises a sustained and controlled release preparation.
Further, the sustained and controlled release preparation comprises a nutritional factor and/or a drug.
In the invention, the release of lipopolysaccharide-induced cell inflammatory factors is inhibited by radio stimulation with a certain frequency, so that the transformation of cell morphology is limited, and the anti-inflammatory aim is achieved. Or combined with sustained and controlled release, a certain amount of electric stimulation is applied, and the system absorbs the medicines such as imidazoline, resveratrol, asiaticoside and the like, inhibits the release of cell inflammatory factors induced by lipopolysaccharide, and achieves the purpose of anti-inflammation.
Sustained and controlled release: the three-dimensional cell scaffold has good conductivity, and the system can be correspondingly changed under the action of an external electric field. The system can relax and absorb specific medicines or nutritional factors by applying radio stimulation with a certain frequency, stop the electrical stimulation or apply electrical stimulation with another frequency, and suddenly start to shrink to release the absorbed medicines or nutritional factors, thereby realizing the sustained and controlled release of the medicines or nutritional factors.
In the invention, the topological parameters and the morphological size of the three-dimensional annular cell scaffold influence the adhesion, migration, proliferation and differentiation of the MSC, and the topological structure enhances the adhesion of the MSC on the scaffold; influence the migration of MSC and increase single cell polarization; the proliferation of MSC is promoted, the stem property of the cells is not affected, and the multi-directional differentiation potential still exists; enhancing differentiation potential mechanism, under different induction conditions, promoting MSC to differentiate into various tissue cells such as fat, bone, cartilage, muscle, tendon, ligament, nerve, liver, cardiac muscle, endothelium, etc.;
topology parameters: topology of crystalline materials, such as: hexagonal lattice, cubic cage type and the like are obtained through adjustment of PH value, temperature and other parameters in the material preparation process; morphology size: refers to the final obtaining of the macroscopic structure of the annular cell scaffold, the size of the annular inner and outer diameters. Different topologies and morphologies can have different effects on the MSC.
By inoculating MSC on a three-dimensional annular cell scaffold (graphene, conductive hydrogel, conductive fiber and conductive aerogel), cell behaviors are regulated and controlled by combining induced current generated by radio stimulation and physicochemical properties of the scaffold. The cells cultured by the method can keep activity and dryness, can reduce transplantation damage, promote MSC directional differentiation efficiency, enhance calcium ion activity, show high synchronism, promote ion channel change, enhance network electrical signals, support functional cell loop growth and promote cell network formation; the technology enhances the plasticity of cells and enhances the spatial memory process of the cells; the technology affects the interaction between the three-dimensional annular cell scaffold and cells, has different effects on inflammation induced by Lipopolysaccharide (LPS), restricts the morphological transformation of cells through the unique characteristics of the technology, and shows anti-inflammatory capability on external injury; the technology stimulates the expression of exosomes, participates in cell communication, promotes cell migration, differentiation, antigen presentation, organism immune response and the like; the technology is combined with sustained and controlled release, releases nutritional factors, controls the release speed, concentration, time and the like of the medicine, acts on cells, reduces toxic and side effects, and regulates and controls the cell behavior.
And (3) common regulation: the radio stimulation parameters are regulated to generate currents with different magnitudes, the electric conductivities generated by different materials and different proportions of the same materials are different, and different influences are generated on cells.
The technical scheme of the present invention is further described in detail below with reference to several preferred embodiments and the accompanying drawings, and the embodiments are implemented on the premise of the technical scheme of the present invention, and detailed implementation manners and specific operation procedures are given, but the protection scope of the present invention is not limited to the following embodiments.
The experimental materials used in the examples used below, unless otherwise specified, are all commercially available from conventional biochemical reagent companies.
Example 1
The copper/nickel template with controllable structure is prepared by adopting any micro-nano processing means of photoetching, plasma etching and electroplating, then the three-dimensional annular copper/nickel template with controllable inner and outer diameters is used as a catalyst, and the three-dimensional annular graphene is grown in a horizontal tubular CVD furnace. Firstly, sequentially placing copper/nickel in absolute ethyl alcohol and deionized water for ultrasonic treatment for 15min to remove surface impurities, sequentially immersing in 0.1mol/L dilute hydrochloric acid for 2min and deionized water for 5min for further cleaning, and drying with nitrogen. At low flow rate H 2 And heating the template to 950 ℃ under high flow rate Ar gas, at high flow rate H 2 And low flow Ar gas for 10min, and then exposed to low flow H 2 High flow Ar and CH 4 The next 5min, finally the CVD system was cooled to room temperature. And placing the template growing with the graphene in ferric chloride corrosive liquid until the template is corroded completely. Sequentially placing the obtained sample in 1mol/L, 0.1mol/L, 0.01mol/L dilute hydrochloric acid and deionized water for cleaning, and then gradually dehydrating and freeze-drying in graded ethanol to prepare the three-dimensional graphene annular cell support; the three-dimensional annular cell scaffold prepared from graphene with a silicon wafer as a substrate is detected by using an energy dispersion X-ray energy spectrometer, so that the graphene is only composed of carbon elements (figure 1). Raman spectra show characteristic peaks of graphene (fig. 2): the D peak near 1350 represents a defect in the carbon lattice, and the sharp G peak near 1580 is represented by sp 2 The 2D peak near 2700 is caused by the resonance of the binaural lattice due to the planar vibration of the orbital hybridization carbon atoms, the intensity ratio of the 2D peak to the G peak is the judgment basis of the number of graphene layers, the ratio is about 0.8, which indicates that the number of graphene layers is 3Left and right.
Planting mesenchymal stem cells on a graphene bracket (or conductive hydrogel, conductive fiber and conductive aerogel bracket), leading sinusoidal alternating current signals to a primary coil, performing wireless electric stimulation on MSC on the bracket by using induction current generated by the change of magnetic flux, and detecting the influence of the electric stimulation and the physicochemical properties of the bracket on MSC differentiation after 7 days of differentiation, as shown in figure 3; fig. 4a-4b show that when sinusoidal ac signals of 2a,500Hz (4 a) and 1000Hz (4 b) are respectively present in the primary coil, induced electromotive forces with amplitudes of 50mV and 100mV, frequencies of 500Hz and 1000Hz and similar to the sinusoidal ac signals are generated on the support, and the induced electromotive forces formed on the support conform to faraday's law of electromagnetic induction (e=nΔΦ/Δt), i.e. the magnitude of the induced electromotive forces is positively linearly related to the number of turns of the coil and the rate of change of the magnetic flux.
FIGS. 5a-5c represent the effect of radio stimulation on cell differentiation tested seven days after cell differentiation: FIG. 5a is a graph showing the effect of radio stimulation (wireess ES) on neuronal dendritic spine density assessed 7 days after differentiation by Tuj-1 (neuronal marker), F-actin (F-actin), DAPI (nuclear marker), merge (fusion of the three markers) staining tests; FIG. 5b is an enlarged view of a portion of FIG. 5 a; the statistics of fig. 5c show that radio stimulation increased the dendritic spine density from 3.28±0.31 spine/10 microns to 4.30±0.25 spine/10 microns during cell differentiation, indicating that radio stimulation plays a role in neurite formation.
Example 2
The preparation of the three-dimensional annular cell scaffold by adopting the 3D printing method comprises the following steps: the 3D printing is one kind of fast forming technology, and is one kind of technology of constructing object with powdered metal, plastic or other adhesive material based on digital model file in layer-by-layer printing mode. The steps are as follows: 1. modeling: manually modeling, and designing a three-dimensional annular structure with controllable inner and outer diameters by using 3D-MAX, Z-Brush and other software; 3D scanning, namely scanning the three-dimensional annular support template by a 3D scanner to generate a digital file. Slicing: and converting the built 3D digital model into a running path which can be identified by a 3D printer and the extrusion amount of consumable materials, loading the model into software, clicking a model slice, and sending a file to the 3D printer after the model slice is finished. 3. Printing: the file is found and the printing is started by clicking. The device was placed on a 3D printer and the central container of the device was filled with the required amount of hydrogel (hydrogel including graphene and polysaccharide).
Characterization of the properties: FIGS. 6a-6b are scanning electron microscope images of a three-dimensional hydrogel annular cell scaffold prepared in example 2 of the present invention; FIG. 7 is a chart showing pore size statistics of a three-dimensional hydrogel annular cell scaffold prepared in example 2 of the present invention; fig. 8a-8c are diagrams of biocompatibility of the three-dimensional hydrogel annular cell scaffold prepared in example 2 of the present invention, wherein fig. 8a is a live cell diagram, fig. 8b is a dead cell diagram, and fig. 8c is a fusion diagram of cells and the three-dimensional hydrogel annular cell scaffold, and it can be seen that the three-dimensional hydrogel annular cell scaffold prepared in this example has good biocompatibility.
In addition, the inventors have also conducted experiments with other materials, process operations, and process conditions described in this specification with reference to the foregoing examples, and all obtained desirable results.
The various aspects, embodiments, features and examples of the invention are to be considered in all respects as illustrative and not intended to limit the invention, the scope of which is defined solely by the claims. Other embodiments, modifications, and uses will be apparent to those skilled in the art without departing from the spirit and scope of the claimed invention.
The use of headings and chapters in this disclosure is not meant to limit the disclosure; each section may apply to any aspect, embodiment, or feature of the present invention.
Throughout this disclosure, where a composition is described as having, comprising, or including a particular component, or where a process is described as having, comprising, or including a particular process step, it is contemplated that the composition of the teachings of the present invention also consist essentially of, or consist of, the recited component, and that the process of the teachings of the present invention also consist essentially of, or consist of, the recited process step.
It should be understood that the order of steps or order in which a particular action is performed is not critical, as long as the present teachings remain operable. Furthermore, two or more steps or actions may be performed simultaneously.
While the invention has been described with reference to an illustrative embodiment, it will be understood by those skilled in the art that various other changes, omissions and/or additions may be made and substantial equivalents may be substituted for elements thereof without departing from the spirit and scope of the invention. In addition, many modifications may be made to adapt a particular situation or material to the teachings of the invention without departing from the scope thereof. Therefore, it is intended that the invention not be limited to the particular embodiment disclosed for carrying out this invention, but that the invention will include all embodiments falling within the scope of the appended claims. Moreover, unless specifically stated any use of the terms first, second, etc. do not denote any order or importance, but rather the terms first, second, etc. are used to distinguish one element from another.
Claims (6)
1. A method of culturing mesenchymal stem cells, comprising:
preparing a copper/nickel template with a controllable structure by adopting at least any micro-nano processing means of photoetching, plasma etching and electroplating, growing graphene on the copper/nickel template by adopting a chemical vapor deposition method, and then performing corrosion, cleaning and drying treatment to prepare the three-dimensional annular cell bracket;
and inoculating the mesenchymal stem cells to the three-dimensional annular cell scaffold, and then carrying out radio stimulation on the three-dimensional annular cell scaffold inoculated with the mesenchymal stem cells for one period by using a primary coil with an electric signal, so as to promote the expression of exosomes and promote the differentiation of the cells into nerve cells; wherein the electrical signal is a sinusoidal alternating current signal: the frequency is 20kHz, the current is 2A, and the stimulation duration is as follows: strings are 1s long, strings are spaced 2s apart, 1h a day, and one cycle is 7 days.
2. The culture method according to claim 1, wherein: the three-dimensional annular cell scaffold also comprises a sustained and controlled release preparation; the sustained and controlled release preparation is selected from nutritional factors and/or medicines.
3. The culture method according to claim 1, wherein the chemical vapor deposition method comprises:
providing a three-dimensional annular copper/nickel template as a catalyst and placing the three-dimensional annular copper/nickel template in a reaction chamber of chemical vapor growth equipment;
introducing hydrogen and argon into the reaction chamber, and carrying out annealing treatment for 10-15min at 950 ℃;
and introducing mixed gas of hydrogen, argon and methane into the reaction chamber, growing a graphene layer on the surface of the three-dimensional annular copper/nickel template by a chemical vapor deposition method, and then carrying out etching treatment to obtain the three-dimensional annular cell support.
4. A culture method according to claim 3, wherein the chemical vapor deposition method further comprises: and cleaning the three-dimensional annular copper/nickel template, drying and then placing the three-dimensional annular copper/nickel template in the reaction chamber.
5. A culture method according to claim 3, wherein the chemical vapor deposition method further comprises: and heating the three-dimensional annular copper/nickel template in the reaction chamber to 950 ℃ under the low-flow hydrogen and high-flow argon atmosphere, and then annealing for 10-15min under the high-flow hydrogen and low-flow argon atmosphere.
6. The method of claim 1, wherein the three-dimensional toroidal cell scaffold has a macroscopic inner diameter of 4-9mm and an outer diameter of 14-20mm.
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